Pharmaceutical Research, Vol. 9, No. I , 1992
`
`
`
`
`
`
`
`
`Review
`
`
`
`
`
`
`
`
`Drug Metabolism in the Nasal Mucosa
`
`
`
`
`
`Mohamadi A. Sarkarl
`
`
`
`
`
`Nasal delivery is a potential alternative for systemic availability of drugs restricted to intravenous
`
`
`
`
`
`
`
`
`
`
`
`
`
`administration, such as peptide and protein drugs. Although nasal delivery avoids the hepatic first-
`
`
`
`
`
`
`
`
`
`
`
`
`
`pass effect, the enzymatic barrier of the nasal mucosa creates a pseudo-flrst—pass eflect. The xeno-
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`biotic metabolic activity in the nasal epithelium has been investigated in several species including
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`humans. The Phase I, cytochrome P-450 enzymes have been studied extensively for their toxicological
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`significance, since these enzymes metabolize inhaled pollutants into reactive metabolites which may
`
`
`
`
`
`
`
`
`
`
`
`
`induce nasal tumors. The cytochrome P-450 activity in the olfactory region of the nasal epithelium is
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`higher even than in the liver, mainly because of a three- to fourfold higher NADPH—cytochrome P-450
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`reductase content. Phase II activity has also been found in the nasal epithelium. The delivery of
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`peptides and proteins has been hindered by the peptidase and protease activity in the nasal mucosa.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`The predominant enzyme appears to be aminopeptidase among other exopeptidases and endopepti-
`
`
`
`
`
`
`
`
`
`
`
`dases. The absorption of peptide drugs can be improved by using aminoboronic acid derivatives,
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`amastatin, and other enzyme inhibitors as absorption enhancers. It is possible that some of the
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`surfactants, e.g., bile salts, increase absorption by inhibiting the proteolytic enzymes. Thus, in addi-
`
`
`
`
`
`
`
`
`
`
`
`
`
`tion to the permeation barriers, there also exists an enzymatic barrier to nasal drug delivery, which is
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`created by metabolic enzymes in the nasal epithelium.
`
`
`
`
`
`
`
`
`KEY WORDS: nasal metabolism; nasal cytochrome P-450; enzyme inhibitors; absorption enhancers.
`
`
`
`
`
`
`
`
`
`
`
`
`
`INTRODUCTION
`
`the nasal mucosa are considered, a brief review of the nasal
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`anatomy and physiology is neceSSary'
`
`
`
`
`Until recently antibiotics antiinflammatory steroids
`
`
`
`
`
`and decongestants were administered intranasally only for
`
`
`
`
`
`
`
`
`
`
`
`
`
`their local action, e.g., nasal decongestion and bronchodila- A“at°mY and PhY5l°l°3Y 0f the N059
`
`
`
`
`
`
`
`tion. The observation that systemic side effects appeared in
`
`
`
`
`
`
`
`
`
`some cases led to the conclusion that the nasal mucosa per-
`
`
`
`
`
`
`
`
`
`
`mits the systemic availability of some drugs. Nasal delivery
`
`
`
`
`
`
`
`
`
`offers a promising alternative to parenteral administration of
`
`
`
`
`
`
`
`drugs that cannot tolerate the rigorous gastrointestinal envi-
`
`
`
`
`
`
`
`
`ronment after oral administration. Nasal administration is
`
`
`
`
`
`
`
`being actively investigated as one of the possible noninva-
`
`
`
`
`
`
`
`
`sive alternatives to delivering peptide and protein drugs.
`
`
`
`
`
`
`
`
`The nasal epithelium has a defensive enzymatic barrier
`
`
`
`
`
`
`
`
`against the entry of xenobiotics, and the original concept of
`
`
`
`
`
`
`
`
`
`
`nasal delivery without first-pass effect is no longer applica-
`
`
`
`
`
`
`
`
`ble. Although the blood circulation from the nose is not pre-
`
`
`
`
`
`
`
`
`
`
`sented to the liver, the nasal mucosa itself is a barrier to the
`
`
`
`
`
`
`
`
`
`
`
`
`
`direct systemic access of some drugs. Drug metabolism in
`
`
`
`
`
`
`
`
`
`the nasal mucosa is an important consideration not only in
`
`
`
`
`
`
`
`
`
`
`nasal drug delivery, but also for toxicologic implications be-
`
`
`
`
`
`
`
`
`cause of xenobiotic metabolism of inhaled environmental
`
`
`
`
`
`
`pollutants or other volatile chemicals. The Phase I cy-
`
`
`
`
`
`
`
`
`tochrome P-450 enzymes can convert some of the airborne
`
`
`
`
`
`
`
`
`
`chemicals to reactive metabolites which may be involved in
`
`
`
`
`
`
`
`
`
`forming DNA adducts, increasing the risk of carcinogenesis
`
`
`
`
`
`
`
`
`in the nasopharynx and lung. Before the metabolic aspects of
`
`
`
`
`
`
`
`
`
`
`
`
`
`The primary function of the nose is olfaction, but it also
`
`
`
`
`
`
`
`
`
`
`filters airborne particulate and heats and humidifies inspired
`
`
`
`
`
`
`
`air. The external portion of the nose does not have any sig-
`
`
`
`
`
`
`
`
`
`
`
`nificance in drug metabolism. The lateral wall of the internal
`
`
`
`
`
`
`
`
`
`nose contains the turbinates, the draining orifices of the
`
`
`
`
`
`
`
`
`paranasal sinuses, and the nasolacrimal duct. The nasal pas-
`
`
`
`
`
`
`
`
`sageways are extremely convoluted, the turbinates divide
`
`
`
`
`
`
`the air spaces into thin slits only a few millimeters wide, and
`
`
`
`
`
`
`
`
`
`
`
`the surface area of the nasal mucosa is considerably in-
`
`
`
`
`
`
`
`
`
`creased (1). In adult humans, the nasal cavities are covered
`
`
`
`
`
`
`
`
`
`
`by a 2- to 4-mm-thick mucosa (2), the nasal cavity’s volume
`
`
`
`
`
`
`
`
`
`is about 20 ml, and its total surface area is about 180 cm2 (1).
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Ethmoturbinates. This term is often seen in the nasal
`
`
`
`
`
`
`
`
`
`metabolism literature. The ethmoid bone is the principal sup-
`
`
`
`
`
`
`
`
`porting structure of the nasal cavities. The ethmoturbinates
`
`
`
`
`
`
`
`are composed principally of the olfactory mucosa. The 01-
`
`
`
`
`
`
`
`
`factory receptors (Fig. 1) lie in the superior nasal turbinates
`
`
`
`
`
`
`
`
`
`(ethmoturbinates), also called the olfactory region. The 01-
`
`
`
`
`
`
`
`factory epithelium of several species is abundant in cy-
`
`
`
`
`
`
`
`
`tochrome P-450 enzymes and is capable of metabolizing
`
`
`
`
`
`
`
`drugs (3). Below the olfactory region, the membrane con-
`
`
`
`
`
`
`
`
`tains capillaries and pseudostratifled ciliated columnar cells
`
`
`
`
`
`
`with many goblet cells and ducts of Bowman’s glands.
`
`
`
`
`
`
`
`
`
`MOXill0lMrbiflai€S- This lS another term frequently €11-
`
`
`
`
`
`
`countered in literature. The paired maxillae unite to form the
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`‘ School of Pharmacy, West Virginia University, Health Sciences
`
`
`
`
`
`
`
`
`North, Morgantown, West Virginia 26506.
`
`
`
`
`
`
`
`
`
`
`
`
`0724-8741/92/0100-000l$06.S0/0 © 1992 Plenum Publishing Corporation
`
`
`
`
`
`
`
`
`Page 1 of 9
`
` Insys Exhibit 2014
`CFAD v. Insys
`IPR2015-01797
`
`

`
`
`
`
`
`Ollacloly bulb
`
`
`
`
`Olfactory (Bowman's) gland
`
`Ollaclory epnhehum
`
`.,in1'-hurts:-c-n",
`
`Cnbmorm plale ot emmoua Done
`
`Ollactovy (I) nerve
`
`j_,__— Connective IISSUE
`Basal cell
`Supporting cell
`Ollactory cell
`
`Ollactory vesicle
`Ollactory nan (aendme)
`Mucus layer
`Substance belflg smelled
`
`Fig. 1. Schematic representation of the olfactory receptors. (Reproduced from G. J. Tortora, Prin-
`
`
`
`
`
`
`
`
`
`
`
`
`
`ciples of Human Anatomy, Harper & Row, New York, p. 559.)
`
`
`
`
`
`
`
`
`
`
`
`
`part of the lateral wall and floor of the nasal cavity. The
`
`
`
`
`
`
`
`
`
`
`
`maxilloturbinates form the respiratory mucosa; the pseudo-
`
`
`
`
`
`
`stratified ciliated columnar epithelium (Fig. 2) is often called
`
`
`
`
`
`
`
`
`respiratory epithelium. The yellow—brown olfactory epithe-
`
`
`
`
`
`lium usually has substantially higher metabolic capabilities
`
`
`
`
`
`
`than does the pinkish-white respiratory epithelium. Mucus-
`
`
`
`
`
`
`secreting goblet cells are interspersed within the nasal mu-
`
`
`
`
`
`
`
`
`cosa with ciliated cells, while the submucosa is rich in both
`
`
`
`
`
`
`
`
`
`
`serous and mucus glands. There is a continuous flow of mu-
`
`
`
`
`
`
`
`
`
`cus within the portions of the nose lined by ciliated respira-
`
`
`
`
`
`
`
`
`
`
`tory epithelium, as a result of the ciliary activity, most of
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Goblet cell
`
`Serous
`gland
`
`Fig. 2. Schematic representation of the respiratory mucosa consist-
`
`
`
`
`
`
`
`
`ing of the pseudostratified columnar epithelium. This histological
`
`
`
`
`
`
`
`section shows the typical appearance of the nasal mucosa. The sur-
`
`
`
`
`
`
`
`
`
`
`face cells are largely ciliated columnar cells, interspersed with gob-
`
`
`
`
`
`
`
`
`
`let cells. Mucus and serous secreting glands are found in the under-
`
`
`
`
`
`
`
`
`
`
`
`lying loose and vascular submucosa. (Reproduced from K. G. Mar-
`
`
`
`
`
`
`
`
`
`shall and E. L. Attia, Disorders of the Nose and Paranasal Sinuses,
`
`
`
`
`
`
`
`
`
`
`
`
`PSG, Littleton, Mass., p. 9.)
`
`
`
`
`
`
`
`
`which is directed posteriorly toward the nasopharynx. The
`
`
`
`
`
`
`
`area below the epithelium is a highly vasculatized connec-
`
`
`
`
`
`
`
`
`tive tissue region.
`
`
`
`Absorption of drugs across the nasal mucosa results in
`
`
`
`
`
`
`
`
`direct systemic exposure, thus avoiding the first~pass he-
`
`
`
`
`
`
`
`patic metabolism associated with oral administration. How-
`
`
`
`
`
`
`ever, an alternative first-pass effect is created, by the met-
`
`
`
`
`
`
`
`
`
`abolic activity within the nasal mucosa. As the existence of
`
`
`
`
`
`
`
`
`
`
`drug metabolizing enzymes in the nasal mucosa of humans
`
`
`
`
`
`
`
`
`
`has not been definitively established, the information pro-
`
`
`
`
`
`
`
`vided in this article relates to different animal species. Me-
`
`
`
`
`
`
`
`
`
`tabolism in the nasal mucosa has been investigated in rat,
`
`
`
`
`
`
`
`
`
`rabbit, Syrian hamster, dog, and monkey (3). It would be
`
`
`
`
`
`
`
`
`
`reasonable to assume that despite some interspecies varia-
`
`
`
`
`
`
`
`tion seen in the nasal drug metabolizing enzymes, humans
`
`
`
`
`
`
`
`
`may also have a similar profile of enzymes in the nasal mu-
`
`
`
`
`
`
`
`
`
`
`
`cosa. Such a comparison can be justified by a report on
`
`
`
`
`
`
`
`
`
`
`metabolism by human nasal microsomes (4), where the re-
`
`
`
`
`
`
`
`
`sults were comparable to the observations in animals (5).
`
`
`
`
`
`
`
`
`
`This speculation is supported by the vast information avail-
`
`
`
`
`
`
`
`
`able on drug metabolizing enzymes in the liver and the fact
`
`
`
`
`
`
`
`
`
`
`that, despite some interspecies differences, most of the he-
`
`
`
`
`
`
`
`
`patic enzymes studied in animals are homologous in humans.
`
`
`
`
`
`
`
`
`
`The various drug metabolizing enzymes identified in the
`
`
`
`
`
`
`
`
`nasal mucosa are oxidative Phase I enzymes, conjugative
`
`
`
`
`
`
`
`Phase II enzymes, and proteolytic enzymes.
`
`
`
`
`
`
`OXIDATIVE PHASE I ENZYMES
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Cytochrome P-450
`
`
`The nasal epithelium is recognized as a potential first
`
`
`
`
`
`
`
`
`
`line of defense of the lung against airborne xenobiotics. It is
`
`
`
`
`
`
`
`
`
`
`
`Page 2 of 9
`
`

`
`Nasal Metabolism
`
`
`
`
`
`
`
`
`The presence of cytochrome P-450-dependent monoox-
`
`
`
`
`
`ygenases has been reported in the nasal tissue of rabbit,
`
`
`
`
`
`
`
`
`
`
`guinea pig, rat, Syrian hamster, mice, and dog (Table I). The
`
`
`
`
`
`
`
`
`
`
`
`highest microsomal cytochrome P-450 concentration was
`
`
`
`
`
`
`found in the Syrian hamster nasal epithelium, and in general,
`
`
`
`
`
`
`
`
`
`
`for all the species the highest microsomal cytochrome P-450
`
`
`
`
`
`
`
`
`
`concentration was found in the ethmoturbinates (olfactory
`
`
`
`
`
`
`
`region). Subsequently the catalytic activity (e.g., aminopy-
`
`
`
`
`
`
`rine N-demethylation) is more pronounced in the mucosa of
`
`
`
`
`
`
`
`
`the olfactory region than in the mucosa covering the respi-
`
`
`
`
`
`
`
`
`
`ratory region (18). In another study (17) catalytic activity,
`
`
`
`
`
`
`
`
`
`using four substrates, was compared between the liver and
`
`
`
`
`
`
`
`
`
`the nasal olfactory epithelium (Table II). All four substrates
`
`
`
`
`
`
`
`
`
`were metabolized more rapidly in the olfactory epithelium,
`
`
`
`
`
`
`
`
`probably because of higher NADPH cytochrome P-450 re-
`
`
`
`
`
`
`
`_ductase activity, which was two- to threefold higher in the
`
`
`
`
`
`
`
`
`olfactory epithelium than the liver. This observation was
`
`
`
`
`
`
`
`consistent across various species and sex (17).
`
`
`
`
`
`
`
`All three major classes of hepatic inducible cytochrome
`
`
`
`
`
`
`
`P-450 isozymes have been found in the nasal mucosa. The
`
`
`
`
`
`
`
`
`
`phenobarbital (PB)-inducible P-450 isozymes are concen-
`
`
`
`
`
`trated within the respiratory epithelium, whereas the 3-me-
`
`
`
`
`
`
`
`thylcholanthrene (MC)—inducible P-450 isozymes are located
`
`
`
`
`
`in the Bowman’s gland of the olfactory region. The preg-
`
`
`
`
`
`
`
`
`
`nenolone-16o(-carbonitrile (PCN)-inducible P-450 isozymes
`
`
`
`
`are distributed uniformly throughout the nasal mucosa (19).
`
`
`
`
`
`
`
`
`Two unique cytochrome P-450 isozymes, NMa and NMb,
`
`
`
`
`
`
`
`
`related to P-450 3a, have also been purified and character-
`
`
`
`
`
`
`
`
`
`ized in the rabbit nasal microsomes (20). Oxidative metabo-
`
`
`
`
`
`
`
`
`lism is thought to play an important role in olfaction.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Drug Metabolism in Human Nasal Mucosa
`
`
`
`
`
`
`Few reports are available on human nasal metabolism
`
`
`
`
`
`
`
`capabilities (4,5). However, these observations strongly sug-
`
`
`
`
`
`
`gest that significant metabolic activity is present in the hu-
`
`
`
`
`
`
`
`
`
`man nasal mucosa. The volatile diethylnitrosamine (DEN)
`
`
`
`
`
`
`present in air, water, foods, and tobacco smoke is metabo-
`
`
`
`
`
`
`
`
`
`lized upon inhalation and, thus, presents a high risk of nasal
`
`
`
`
`
`
`
`
`
`tumors to individuals working in the leather or wood indus-
`
`
`
`
`
`
`
`
`
`try (21). Investigation of catalytic activity (4) showed that
`
`
`
`
`
`
`
`
`the DEN-deethylase activity of human nasal mucosa mi-
`
`
`
`
`
`
`
`crosomes was 1.06 : 0.99 nmol/mg/min (n = 4) and that of
`
`
`
`
`
`
`
`
`
`
`
`human liver microsomes was 2.7 : 1.44 nmol/mg/min (n =
`
`
`
`
`
`
`
`
`
`
`5). Since the P-450 content in the human liver is 0.2-0.8
`
`
`
`
`
`
`
`
`
`
`nmol/mg protein (22) and 0.026 nmol/mg protein in the hu-
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Table I. Cytochrome P-450 in Nasal Mucosa“
`
`
`
`
`
`
`
`Conc. cytochrome P-450
`
`
`
`(pmol P-450/mg microsomal protein)
`
`
`
`Total nasal tissue”
`
`
`
`460 : s (12)
`
`
`
`
`420
`350 -_- 71 (3)
`
`
`
`235 -_- 43 (3)
`
`
`
`
`110 -5 15 (9)
`
`
`
`
`94 : 6 (12)
`
`
`
`
`as : 5 (30)
`
`
`
`
`
`Animal
`
`
`Syrian hamster
`
`
`Cynomolgus monkey
`
`Rabbit
`
`Dog
`
`Rat
`
`Guinea pig
`
`
`Mouse
`
`“ Data presented from Ref. 3.
`
`
`
`
`
`
`" Number of animals in parentheses; results are mean 1' SE.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`constantly exposed to the external environment and is one of
`
`
`
`
`
`
`
`
`
`the major target organs for toxicity of many inhaled sub-
`
`
`
`
`
`
`
`
`
`stances. For example, many environmental pollutants ad-
`
`
`
`
`
`
`sorbed onto particles (of sizes 1- to l00—p.m mass median
`
`
`
`
`
`
`
`
`aerodynamic diameter) have been observed to deposit in the
`
`
`
`
`
`
`
`
`nasopharyngeal region of the respiratory tract (6).
`
`
`
`
`
`
`
`Studies of biotransformation of foreign compounds in
`
`
`
`
`
`
`
`the nasal mucosa of animals have shown extensive P-450-
`
`
`
`
`
`
`
`
`dependent metabolism. Some cytochrome P-450 enzymes
`
`
`
`
`
`
`have been linked to increased risk of cancer and/or other
`
`
`
`
`
`
`
`
`
`
`toxicological effects such as lesions in the nasal mucosa,
`
`
`
`
`
`
`
`
`
`because of formation of reactive metabolites which can form
`
`
`
`
`
`
`
`
`
`mutagenic DNA adducts. Nasal cancer has been reported to
`
`
`
`
`
`
`
`
`
`occur in experimental animals after inhalation exposure to a
`
`
`
`
`
`
`
`
`variety of important industrial chemicals, including formal-
`
`
`
`
`
`
`dehyde (at an exposure of 15 ppm) (7), hexamethylphos-
`
`
`
`
`
`
`
`
`phoramide (8), and the tobacco—specific nitrosamines (9).
`
`
`
`
`
`
`
`The common air pollutant, benzo(a)pyrene (BaP), has been
`
`
`
`
`
`
`
`
`shown to be converted to the carcinogenic metabolite BaP-
`
`
`
`
`
`
`
`
`7,8-diol-9,10-epoxide in the rat nasal tissue (10). Inhalation
`
`
`
`
`
`
`
`
`studies with benzo(a)pyrene in Syrian hamsters resulted in
`
`
`
`
`
`
`
`
`neoplasms in the nasal cavity, larynx, pharynx, esophagus,
`
`
`
`
`
`
`
`
`and forestomach (11). The gastrointestinal tract tumors
`
`
`
`
`
`
`
`could possibly be a result of swallowing of the mucus laden
`
`
`
`
`
`
`
`
`
`
`
`with BaP or its reactive metabolites. Nasal administration is
`
`
`
`
`
`
`
`
`
`not necessary to initiate the carcinogenic effect of P-450 me-
`
`
`
`
`
`
`
`
`
`tabolism. Long-term experiments have indicated that oral
`
`
`
`
`
`
`administration of large doses of phenacetin to rats will in-
`
`
`
`
`
`
`
`
`
`duce carcinomas of the nose (12).
`
`
`
`
`
`
`Numerous other compounds have been shown to be me-
`
`
`
`
`
`
`
`
`tabolized in vitro by the nasal P—450—dependent monooxy—
`
`
`
`
`
`
`
`genase system, e.g., nasal decongestants, essences, anes-
`
`
`
`
`
`
`thetics, alcohols, nicotine, and cocaine (13). A number of
`
`
`
`
`
`
`
`
`
`these compounds are also metabolized in vivo in the nasal
`
`
`
`
`
`
`
`
`
`
`mucosa, as demonstrated by whole-body autoradiography
`
`
`
`
`
`
`(14). The results from these studies indicate that the nasal
`
`
`
`
`
`
`
`
`
`
`mucosa is, in many cases, much more active (on a per mil-
`
`
`
`
`
`
`
`
`
`
`
`ligram of protein basis) than other organs, including liver, in
`
`
`
`
`
`
`
`
`
`
`the metabolism of foreign compounds both in vitro and in
`
`
`
`
`
`
`
`
`
`
`vivo.
`
`The specific content of the F-450 in nasal mucosa is
`
`
`
`
`
`
`
`
`
`
`relatively high, second only to that of the liver. The most
`
`
`
`
`
`
`
`
`
`
`
`striking feature is that the catalytic activity of the P-450
`
`
`
`
`
`
`
`
`
`
`enzymes in the nasal epithelium is higher than in any other
`
`
`
`
`
`
`
`
`
`
`
`tissue including the liver. The relatively high levels of the
`
`
`
`
`
`
`
`
`
`
`cytochrome P-450 enzymes and the maximal catalytic activ-
`
`
`
`
`
`
`
`ity serve as a protective mechanism against the constant
`
`
`
`
`
`
`
`
`
`barrage of xenobiotics. The higher activity could be a result
`
`
`
`
`
`
`
`
`
`of a higher NADPH—cytochrome P-450 reductase content,
`
`
`
`
`
`
`which is the rate—limiting factor in the cytochrome P-450
`
`
`
`
`
`
`
`
`
`oxidoreductase cycle, since the reductase is involved in the
`
`
`
`
`
`
`
`
`
`transfer of the first or the second electron in the cycle (15).
`
`
`
`
`
`
`
`
`
`
`
`
`Immunoblot analysis with antireductase IgG has shown that
`
`
`
`
`
`
`
`
`rabbit nasal microsomes have very high levels of cy-
`
`
`
`
`
`
`
`
`tochrome P-450 reductase (16). The ratio of NADPH-
`
`
`
`
`
`
`
`cytochrome P-450 reductase to cytochrome P-450 content is
`
`
`
`
`
`
`
`
`1:11 to 1:15 in the liver, whereas in the olfactory region of
`
`
`
`
`
`
`
`
`
`
`
`
`the nasal epithelium this ratio is 1:2 to 1:3 (17). The higher
`
`
`
`
`
`
`
`
`
`
`
`
`proportion of NADPH reductase makes the cytochrome
`
`
`
`
`
`
`
`P-450 enzymes more efficient, thereby considerably increas-
`
`
`
`
`
`
`ing the catalytic activity.
`
`
`
`
`
`Page 3 of 9
`
`

`
`
`
`Liver
`
`
`
`Olfactory
`
`epithelium
`
`
`
`
`
`
`
`
`
`
`4 T
`
`
`
`able II. Drug Metabolism in Microsomes Isolated from Liver and
`
`
`
`
`
`
`
`
`
`Olfactory Epithelium of Male Hamsters”
`
`
`
`
`
`Metabolic activity
`
`
`(nmol/mg protein/min)
`
`Cytochrome P-450
`
`
`1.15 1 0.19
`0.58 : 0.14
`(nmol/mg protein)
`
`
`
`
`
`
`27.10 : 11.9
`3.74 I 0.58
`7-Ethyoxycoumarin deethylase
`
`
`
`
`
`0.56 : 0.10
`0.21 : 0.05
`7—Ethoxyresoruf1n deethylase
`
`
`
`
`
`
`
`45.9
`1.4
`Hexobarbitone oxidase
`
`
`
`
`4.67 : 0.73
`0.95 : 0.15
`Aniline hydroxylase
`
`
`
`
`
`
`
`
`“ Data reproduced from Ref. 17. Results are mean 1 SD of at least
`
`
`
`
`
`
`
`
`
`
`
`
`
`three observations, each from tissues pooled from at least two
`
`
`
`
`
`
`
`
`
`animals. Hexobarbitone oxidase results are means of two obser-
`
`
`
`
`
`
`
`
`vations.
`
`
`
`
`
`
`
`
`
`Epoxide Hydroxylase
`
`
`The reactive epoxides generated by the cytochrome
`
`
`
`
`
`
`P-450 enzymes are inactivated by hydrolysis, hence it is not
`
`
`
`
`
`
`
`
`
`surprising to find epoxide hydroxylase in olfactory and re-
`
`
`
`
`
`
`
`
`spiratory epithelia, Bowman’s glands, and seromucous
`
`
`
`
`
`
`glands, the respiratory region having the highest levels of
`
`
`
`
`
`
`
`
`
`epoxide hydroxylase. The epoxide hydroxylase activity of
`
`
`
`
`
`
`
`rat nasal tissue tends to reflect that reported for rat liver
`
`
`
`
`
`
`
`
`
`
`
`(10,27).
`
`
`
`
`
`Carboxylesterase
`
`
`These enzymes can be of special importance for deliv-
`
`
`
`
`
`
`
`
`ery of drugs with carboxylic acid esters as functional groups.
`
`
`
`
`
`
`
`
`
`
`The interest in this enzyme is generated because of the de-
`
`
`
`
`
`
`
`
`
`
`velopment of lesions in the nasal mucosa after exposure to
`
`
`
`
`
`
`
`
`
`certain volatile industrial esters, e.g., solvents used in paint
`
`
`
`
`
`
`
`
`and coating industries. The ability of rodent nasal mucosa to
`
`
`
`
`
`
`
`
`
`hydrolyze these and a series of other esters to acid and al-
`
`
`
`
`
`
`
`
`
`
`
`cohol metabolites has been studied (28). For certain sub-
`
`
`
`
`
`
`
`
`strates, the rate of hydrolysis is equivalent to or greater than
`
`
`
`
`
`
`
`
`
`
`
`that found when using tissue preparations from liver (29).
`
`
`
`
`
`
`
`
`
`The activity of olfactory mucosal carboxylesterases has
`
`
`
`
`
`
`
`been shown to be three to six times greater than respiratory
`
`
`
`
`
`
`
`
`
`
`
`mucosa when p-nitrophenyl butyrate is used as a substrate
`
`
`
`
`
`
`
`
`
`(29).
`
`
`
`
`
`
`Carbonic Anhydrase
`
`
`Carbonic anhydrase, the zinc-containing enzyme that
`
`
`
`
`
`accelerates the rate of formation of carbon dioxide from car-
`
`
`
`
`
`
`
`
`
`bonic acid, is present at a high concentration in rat olfactory
`
`
`
`
`
`
`
`
`
`cells but absent in supporting cells. Besides its normal func-
`
`
`
`
`
`
`
`
`
`tion with carbonic acid as a substrate, carbonic anhydrase
`
`
`
`
`
`
`
`
`exhibits considerable simple esterase activity and may be
`
`
`
`
`
`
`
`responsible for a major part of the olfactory esterase activity
`
`
`
`
`
`
`
`
`(30).
`
`
`
`
`
`
`
`
`
`
`CONJUGATIVE PHASE II ENZYMES
`
`
`
`
`Comparative evaluation of nasal metabolism of 178-
`
`
`
`
`
`
`estradiol indicated that significantly more conjugation oc-
`
`
`
`
`
`
`curred when the drug was administered via the nasal route
`
`
`
`
`
`
`
`
`
`
`compared with the iv route (31). The extent of conjugation
`
`
`
`
`
`
`
`
`
`
`following nasal administration of 17B-estradiol decreased as
`
`
`
`
`
`
`
`the dose increased, suggesting that the drug is conjugated
`
`
`
`
`
`
`
`
`
`within the nasal mucosa and that this process is saturable.
`
`
`
`
`
`
`
`
`
`
`The authors did not determine the specific conjugated me-
`
`
`
`
`
`
`
`
`tabolites, hence it is not possible to evaluate which of the
`
`
`
`
`
`
`
`
`
`
`conjugation pathways is saturated.
`
`
`
`
`
`
`
`Glucuronyl and Sulfate Transferase
`
`
`
`
`Studies utilizing pooled nasal tissues from the maxilla-
`
`
`
`
`
`
`
`turbinates, ethmoturbinates, and nasal epithelial membrane
`
`
`
`
`
`has shown the presence of these enzymes (32).
`
`
`
`
`
`
`
`
`
`Glutathione Transferase (GST)
`
`
`
`This is a Phase II detoxifying enzyme converting the
`
`
`
`
`
`
`
`
`electrophilic reactive metabolites, formed by Phase I en-
`
`
`
`
`
`
`
`zymes, into harmless glutathione conjugates. Thus, GST is
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`man respiratory nasal mucosa (5), the DEN-deethylation ac-
`
`
`
`
`
`
`
`tivity of nasal mucosa, expressed per nanomole of P-450, is
`
`
`
`
`
`
`
`
`
`
`2-10 times higher than that of human liver.
`
`
`
`
`
`
`
`
`The nasal mucosa, in particular the olfactory region of
`
`
`
`
`
`
`
`
`
`the nasal cavity, is rich in the cytochrome P-450 enzymes
`
`
`
`
`
`
`
`
`
`
`that metabolize xenobiotics. This “first-pass effect” of the
`
`
`
`
`
`
`
`
`nasal mucosa should be taken into considerations for drugs
`
`
`
`
`
`
`
`
`
`to be delivered by the nasal route. The high oxidative met-
`
`
`
`
`
`
`
`
`
`
`abolic capabilities of the nasal mucosa should also be taken
`
`
`
`
`
`
`
`
`
`
`into account in toxicokinetic models intended to predict the
`
`
`
`
`
`
`
`
`
`fate of inhaled compounds.
`
`
`
`
`
`
`
`Steroid Metabolism by Nasal Mucosa
`
`
`
`
`
`Alternative routes of administration of steroids are
`
`
`
`
`
`
`needed especially for progesterone and testosterone, be-
`
`
`
`
`
`
`cause of their poor oral absorption. The nasal bioavailability
`
`
`
`
`
`
`
`
`
`after in vivo administration of the same dose of progesterone
`
`
`
`
`
`
`
`
`
`
`to rats was 100% (relative to iv), compared to only 1.2% for
`
`
`
`
`
`
`
`
`
`
`
`
`intraduodenal bioavailability (23). However, conflicting re-
`
`
`
`
`
`sults have been observed in in vitro studies in the rat nasal
`
`
`
`
`
`
`
`
`
`
`
`mucosa. Extensive metabolism and uptake of progesterone
`
`
`
`
`
`
`and testosterone are noted in the rat nasal mucosa in vitro
`
`
`
`
`
`
`
`
`
`
`(24). The pattern of metabolites shows that these steroids are
`
`
`
`
`
`
`
`
`
`reduced as well as hydroxylated at multiple positions. This
`
`
`
`
`
`
`
`
`discrepancy between the in vitro and the in vivo results could
`
`
`
`
`
`
`
`
`
`
`be due to rapid uptake of progesterone by the respiratory
`
`
`
`
`
`
`
`
`
`mucosa, which is less abundant in metabolizing enzymes
`
`
`
`
`
`
`
`than the olfactory mucosa.
`
`
`
`
`In addition to the cytochrome P-450 enzymes, the fol-
`
`
`
`
`
`
`
`
`lowing oxidative Phase I enzyme activity have also been
`
`
`
`
`
`
`
`
`studied in the nasal epithelium.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Flavin-Containing Monooxygenases and
`
`
`Aldehyde Dehydrogenases
`
`
`The monooxygenase activity is high in the nasal ethmo-
`
`
`
`
`
`
`
`
`turbinates of rabbit respiratory tract in the area of the olfac-
`
`
`
`
`
`
`
`
`
`
`tory mucosa, with levels exceeding those found in the liver
`
`
`
`
`
`
`
`
`
`
`(25). Aldehyde dehydrogenases I and II and formaldehyde
`
`
`
`
`
`
`
`dehydrogenase have also been detected in the nasal cavity of
`
`
`
`
`
`
`
`
`
`
`the rat (26). The specific activities of aldehyde dehydroge-
`
`
`
`
`
`
`
`
`nase II and formaldehyde dehydrogenase in homogenates of
`
`
`
`
`
`
`
`
`olfactory epithelia were higher than in respiratory epithelial
`
`
`
`
`
`
`
`
`homogenates.
`
`
`Page 4 of 9
`
`

`
`Nasal Metabolism
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`important for the inactivation of inhaled mutagens and car-
`
`
`
`
`
`
`
`
`cinogens. Reports indicate that a significant level of GST
`
`
`
`
`
`
`
`
`activity is located in the cytosol of olfactory and respiratory
`
`
`
`
`
`
`
`
`
`mucosa in humans. The specific activity was higher than that
`
`
`
`
`
`
`
`
`
`reported for a number of extrahepatic tissues (33), suggest-
`
`
`
`
`
`
`
`
`ing the potential of nasal mucosa in protection of the body
`
`
`
`
`
`
`
`
`
`
`against the toxic effect of compounds present in the inhaled
`
`
`
`
`
`
`
`
`
`air. The activities of the Phase II enzymes may be dependent
`
`
`
`
`
`
`
`
`
`
`upon the activated cofactor in these tissues (10,27). Quanti-
`
`
`
`
`
`
`
`
`ties of glutathione, on a per gram tissue basis, in rat nasal
`
`
`
`
`
`
`
`
`
`
`
`tissue were found to be about one-tenth those typically re-
`
`
`
`
`
`
`
`
`
`ported in rat liver. Thus, the reactive intermediates formed
`
`
`
`
`
`
`
`
`
`by the cytochrome P-450 enzymes could accumulate when
`
`
`
`
`
`
`
`
`available glutathione is exhausted by conjugation reactions.
`
`
`
`
`
`
`
`The above Phase I and Phase II enzymes have been
`
`
`
`
`
`
`
`
`
`
`studied in the nasal mucosa only for their toxicological im-
`
`
`
`
`
`
`
`
`
`portance, since it is the site for producing toxic metabolites
`
`
`
`
`
`
`
`
`
`
`from the inhalation exposure of environmental pollutants.
`
`
`
`
`
`
`
`This hypothesis seems very likely, since the cytochrome
`
`
`
`
`
`
`
`
`P-450 activity is higher in the nasal mucosa than any other
`
`
`
`
`
`
`
`
`
`
`
`site in the body, even the liver.
`
`
`
`
`
`
`
`This information can be useful when formulating a drug
`
`
`
`
`
`
`
`
`
`for nasal delivery. The earlier theory, that the nasal delivery
`
`
`
`
`
`
`
`
`
`
`eliminates first-pass metabolism in the liver, can be refuted,
`
`
`
`
`
`
`
`
`
`since the nasal mucosa could itself present a first-pass effect.
`
`
`
`
`
`
`
`
`
`The presence of esterases (28) in the nasal mucosa provides
`
`
`
`
`
`
`
`
`
`
`a unique opportunity to avoid the nasal first-pass effect by
`
`
`
`
`
`
`
`
`
`
`designing ester prodrugs for nasal delivery. The product
`
`
`
`
`
`
`
`
`would be rapidly absorbed through the nasal mucosa and
`
`
`
`
`
`
`
`
`
`metabolized by the esterases, and the parent compound
`
`
`
`
`
`
`
`
`would be available in the systemic circulation.
`
`
`
`
`
`
`
`
`PROTEOLYTIC ENZYMES AND THEIR IMPACT ON
`
`
`
`
`
`
`DELIVERY OF PEPTIDE AND PROTEIN DRUGS IN
`
`
`
`
`
`
`
`THE NASAL MUCOSA
`
`
`
`
`
`
`
`
`
`The oral delivery of peptides and proteins has not been
`
`
`
`
`
`
`
`
`
`successful, primarily because of extensive digestion of these
`
`
`
`
`
`
`
`substances by protease and proteinases in the gastrointesti-
`
`
`
`
`
`
`
`nal tract. Consequently, alternative routes, e.g., via the na-
`
`
`
`
`
`
`
`
`sal mucosa, which are presumed to be deficient in these
`
`
`
`
`
`
`
`
`
`enzymes, are being investigated for peptide and protein de-
`
`
`
`
`
`
`
`
`livery. Some peptides can be absorbed in a systemically ef-
`
`
`
`
`
`
`
`
`
`fective quantity following transnasal administration (see Ref.
`
`
`
`
`
`
`
`34 and references therein). On the other hand, variable and
`
`
`
`
`
`
`
`
`
`
`low systemic absorption has also been reported for polypep-
`
`
`
`
`
`
`
`
`tide hormones with a large molecular size, such as insulin
`
`
`
`
`
`
`
`
`
`(34) and leutinizing hormone releasing hormone (35). Al-
`
`
`
`
`
`
`
`though the bioavailability of peptides and proteins from the
`
`
`
`
`
`
`
`
`nasal mucosa is substantially improved over the oral route, it
`
`
`
`
`
`
`
`
`
`is still far from optimal when compared to intravenous
`
`
`
`
`
`
`
`
`routes. This result may be attributed to the resistance en-
`
`
`
`
`
`
`
`
`
`countered by peptides and proteins in penetrating the nasal
`
`
`
`
`
`
`
`
`mucosa, as well as the susceptibility to degradation by pro-
`
`
`
`
`
`
`
`
`
`teases and proteinases that may be present in the mucosa.
`
`
`
`
`
`
`
`
`
`
`The inadequate bioavailability of peptides and proteins even
`
`
`
`
`
`
`
`
`in the presence of penetration enhancers suggests that there
`
`
`
`
`
`
`
`
`
`is another barrier, an enzymatic barrier, which limits absorp-
`
`
`
`
`
`
`
`
`tion. The mucosal membranes of the nasal cavity are known
`
`
`
`
`
`
`
`
`
`
`to have various types of peptidase and protease activities,
`
`
`
`
`
`
`
`
`
`including both exopeptidases and endopeptidases (36). Lee
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`and co-workers (37,38) demonstrated that the proteolytic ac-
`
`
`
`
`
`
`
`
`tivities in homogenates of the mucosal tissues of the albino
`
`
`
`
`
`
`
`
`
`rabbit against methionine enkephalin, [D-Ala2]methionine
`
`
`
`
`enkephalinamide, substance P, insulin, and proinsulin were
`
`
`
`
`
`
`comparable to those in the ileum. On evaluation of the me-
`
`
`
`
`
`
`
`
`
`
`tabolism of the peptides enkephalin, leucine-enkephalin, and
`
`
`
`
`
`
`met-enkephalinamide in homogenates of nasal mucosa,
`it
`
`
`
`
`
`
`was found that all the peptides disappeared rapidly (39), pre-
`
`
`
`
`
`
`
`
`
`sumably because of the action of aminopeptidases, dipepti-
`
`
`
`
`
`
`
`dyl peptidase, and dipeptidyl carboxypeptidase. Based on
`
`
`
`
`
`
`
`the fractions of amino acids obtained after metabolism of
`
`
`
`
`
`
`
`
`
`enkephalins, the authors felt that aminopeptidases were the
`
`
`
`
`
`
`
`
`principal peptidases mediating the hydrolysis of enkepha-
`
`
`
`
`
`
`lins. Thus, inhibition of the aminopeptidase should minimize
`
`
`
`
`
`
`
`enkephalin degradation. The authors cautioned, however,
`
`
`
`
`
`that the structural organization of the peptidases that con-
`
`
`
`
`
`
`
`
`tribute to the hydrolysis of enkephalins and other peptides
`
`
`
`
`
`
`
`
`
`and proteins can be destroyed upon homogenization. It is
`
`
`
`
`
`
`
`
`conceivable that significant differences in the susceptibility
`
`
`
`
`
`
`
`of a given peptide to hydrolysis could be discerned in vivo if
`
`
`
`
`
`
`
`
`
`
`
`the peptidases have different structural organization (39).
`
`
`
`
`
`
`
`In conclusion, aminopeptidase activity in the nasal mu-
`
`
`
`
`
`
`
`cosa has been found to be similar to that of the ileal mucosa
`
`
`
`
`
`
`
`
`
`
`
`
`in its subcellular distribution. Specifically, almost half of the
`
`
`
`
`
`
`
`
`aminopeptidase activity in the nasal mucosa of the albino
`
`
`
`
`
`
`
`
`rabbit is membrane bound (40). It has yet to be determined
`
`
`
`
`
`
`
`
`
`
`whether the membrane-bound aminopeptidases are active
`
`
`
`
`
`enough to de