Toxicological Implications of Nasal Formulations
`
`Muhammad Quadir, Hossein Zia, and Thomas E. Needham
`Department of Pharmaceutics, College of Pharmacy, University of Rhode Island, Kingston,
`Rhode Island, USA
`
`Various nasal formulations have been tested for their suitabil-
`ity to deliver drugs through the nasal cavity. This route is espe-
`cially of interest where the dose of drug is small and the drug may
`undergo an extensive  rst-pass metabolism and/or decomposition
`while passing through the gastrointestinal tract. Unfortunately, the
`nasal mucosa does not have same type of tolerability to all drugs
`and additives used in formulations. Some chemicals may damage
`the nasal epithelia or alter the mucociliary defensive mechanism
`of the nose. There also is a possibility that the drug can transport
`directly from nasal cavity to the brain via the olfactory route. Sev-
`eral methods have been developed to study the impact of drugs and
`excipients on the integrity of the nose. In some cases, the in vitro
`results did not correlate well with in vivo data, due to lack of re-
`producibility of the natural body environment, and some in vitro
`methods may not be sensitive enough and thus may complicate
`interpretation of the results. This review provides a toxicological
`evaluation of different drugs and additives used to optimize a nasal
`formulation. Certain chemicals are now routinely used as additives
`in nasal formulations. Although these compounds are most likely
`safe, if they are used over the long term, they may damage the ep-
`ithelia of the nose. For multidose preparations, preservatives are
`often included in nasal delivery systems and may cause ciliotoxic
`effects. Both physicochemical parameters of drugs as well as for-
`mulation materials should be considered in evaluating the overall
`effect of a drug product on the nose. Therefore, any prior knowledge
`of the effect of drugs and additives on the nasal epithelia ultimately
`will assist in the development of nasal products. Furthermore, as
`the sites of absorption in the nasal cavity are somewhat limited,
`evaluation of the long-term tolerability of a nasal formulation is of
`great importance.
`
`The recent literature reports that administration of certain
`drugs intranasally for systemic effect has proven to be very effec-
`tive. The nasal route is especially advantageous as an alternative
`means for the delivery of drugs that undergo extensive  rst-pass
`metabolism or are sensitive to gastrointestinal decomposition
`(Zia, Dondeti, and Needham 1993). Many small molecules, like
`dihyroergotamine, metaclopramide, butarphanol tartrate, su-
`
`Received 2 February 1999; accepted 10 March 1999.
`Address correspondence to Hossein Zia, Department of Pharma-
`ceutics, College of Pharmacy, University of Rhode Island, Kingston,
`RI 02881, USA.
`
`bistorphanol succinate, and larger molecules such as vitamin
`B12, vasopressin, calcitonin, and even insulin have been suc-
`cessfully delivered intranasally. Although this route has a sig-
`ni cantly higher potential impact on the bioavailability of drugs
`in comparison to other routes, several other factors may in u-
`ence its viability. The mechanism of absorption of drug through
`the nasal cavity has not been fully elucidated.
`A variety of drugs with different physicochemical factors
`are absorbed by the nasal mucosa (Hussain et al. 1980; Su,
`Campanale, and Gries 1984). It seems that neither hydrophobic -
`ity nor hydrophilicity is the sole determining factor for nasal ab-
`sorption. The anatomy of the nasal mucosal barrier suggests that
`several separate compartments may contribute to the permeabil-
`ity of the transnasal passage of drugs. The existence of an aque-
`ous boundary layer also may in uence the transnasal absorption
`of both lipophilic and hydrophilic drugs (Krishnamoorthy and
`Mitra 1998; Roche 1977). Therefore, any change in the complex
`architecture within the nasal passage due to the nasal formula-
`tion may ultimately affect the bioavailability of drugs.
`The nasal epithelium is covered by many hair-like cilia that
`beat in a coordinated manner within the periciliary  uid be-
`neath a layer of viscoelastic mucus. This movement within the
`nose results in mucociliary clearance. After nasal inhalation, mu-
`cociliary clearance contributes to the body’s primary nonspeci c
`defense mechanism by entrapping such potentially hazardous
`materials as dust and microorganisms, allergens, carcinogens,
`and cellular debris within the mucus blanket. The entrapped
`materials are then propelled by the claw-like tips of the under-
`lying cilia toward the pharynx and either swallowed or expec-
`torated (Proctor 1977). Nasal medication should not in uence
`this self-cleaning capacity of the nose. It has been shown that
`the “immotile cilia syndrome” leads to recurrent infections of
`the airways (Afzelius 1979; Duchateau et al. 1985) which is
`linked to the increased occurrence of bronchiestasis, bronchial
`infection, and chronic rhinitis.
`Many drugs and additives have demonstrated inhibition of
`nasal ciliary movement. For instance, ciliostatic agents such as
`mercuric preservatives, antihistamines, dihydroxy bile salts, lo-
`cal anesthetics, and active agents like propranolol, atropine, and
`salmeterol reduce the ciliary beat frequency (Wanner, Salathe,
`and O’Riordan 1996; Kanthakumar et al. 1994; Hermens and
`
`Drug Delivery, 6:227–242, 1999
`Copyright c
`1999 Taylor & Francis
`1071-7544/99 $12.00 + .00
`
`227
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`M. QUADIR ET AL.
`
`Merkus 1987; Rutland, Griffen, and Cole 1982). Therefore, it is
`important to investigate the occurrence of a ciliostatic effect and
`if it is reversible after withdrawal of drug or excipient exposure.
`Any drug or additive used in nasal delivery should be devoid of
`serious ciliotoxicity since the overall feasibility of a nasal drug
`formulation may depend largely on the effects of the ciliated
`epithelial tissue. Although the nose is exposed continuously to
`airborne environmental chemicals as well as those substances
`present in the general circulation, this paper only reviews the
`effects of drugs and additives that have been recently evaluated
`in nasal drug delivery systems.
`
`NOSE
`An extensive description of the nose can be obtained from
`many of the available human anatomy and physiology books.
`The main features relevant to nasal delivery follow. The nostrils
`are a pair of nasal cavities divided by a nasal septum; their to-
`tal volume is approximately 15 cc3, with a total surface area of
`150 cm2. These nasal cavities are covered by a mucosa with a
`thickness of 2–4 mm, whose function in humans is 20% olfac-
`tory and 80% respiratory. The nasal epithelium has a relatively
`high permeability, and only two cell layers separate the nasal lu-
`men from the dense blood vessel network in the lamina propria
`(Pontiroli, Calderara, and Pozza 1989). The human nasal cav-
`ity is lined with three types of epithelia: squamous, respiratory,
`and olfactory (Figure 1). The mucosa in the anterior part of the
`nose is squamous and without cilia. Within the anterior nostrils,
`a transitional epithelium is found that precedes the respiratory
`epithelium. The olfactory epithelium is present in the posterior
`
`FIG. 1. Lateral wall of the nasal cavity. (A) Squamous epithelium, (B) in-
`ferior turbinate, (C) middle turbinate, (D) superior turbinate, (E) frontal sinus,
`(F) respiratory epithelium, (G) olfactory epithelium, (H) sphenoidal sinus, and
`(I) faucial tonsil.
`
`FIG. 2. Respiratory epithelium. (A) Mucus, (B) epithelium, (C) lamina pro-
`pria/submucosa, (D) basal cell, (E) nonciliated cell, (F) microvilli, (G) cilia,
`(H) osmiophyllic membrane, (I) epiphase, (J) hypophase , (K) goblet cell,
`(L) ciliated cell, (M) basement membrane, (N) blood vessel, (O) nerve, and
`(P) gland.
`
`part of the nasal cavity (Emmeline et al. 1995). The epithelium
`contains ciliary cells that produce a unidirectional  ow of mucus
`toward the pharynx. A drug deposited posteriorly in the nose is
`cleared more rapidly from the nasal cavity than a drug deposited
`anteriorly, because clearance is slower at the anterior part of the
`nose than in the more ciliated posterior (Kublik and Vidgren
`1998).
`The respiratory epithelium is the major lining of the human
`nasal cavity (Figure 2) and probably is the primary site for
`systemic absorption of nasally administered drugs (Monteiro-
`Rivera 1984). This epithelium is composed of ciliated and non-
`ciliated columnar cells, goblet cells, and basal cells. The colum-
`nar and goblet cells are found on the apical side of the cell layer
`adjacent to the lumen of the nasal cavity. Basal cells are found
`adjacent to the basal lamina, on the basolateral side of the ep-
`ithelium. The lamina propria is located beneath the basal lamina
`and contains many blood vessels, nerves, and glands.
`
`METHODOLOGY TO EVALUATE NASAL
`FORMULATIONS
`Most of the toxicological studies, both regulatory and aca-
`demic, involve the use of whole animals. Today, we are in u-
`enced by the concept of the three Rs of humane animal use in
`research: replacement (utilization of models that do not involve
`live animals), reduction (use of fewer animals), and re nement
`(minimization of animal suffering). Thus, in vitro systems have
`widespread use for drug development studies (Reed 1997). For
`nasal delivery, several in vitro and in vivo models have been de-
`veloped to study the impact of different drugs and excipients on
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`TOXICOLOGICAL IMPLICATIONS OF NASAL FORMULATIONS
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`229
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`the integrity of the nose. These techniques include mucocilliary
`clearance, morphology of nasal mucosa, biochemical alteration
`or enzyme release, and changes in blood  ow.
`It also has been reported that there is a direct connection of
`nasal mucosa with the cerebrospinal and the central nervous sys-
`tem (Sakane et al. 1997). There is evidence of a phenomena in
`which cerebrospinal  uid leaks through the nasal mucosa to the
`nasal cavity without any underlying causes, when the intracra-
`nial pressure is elevated. Furthermore, an infectious organism
`has been shown to reach the olfactory nerve through the nasal
`mucosa (Tolley and Schwartz 1991). Thus, it is important to
`evaluate the potential impact of a drug formulation on the cen-
`tral nervous system when administering a drug intranasally.
`
`Mucociliary Clearance
`Several pathological conditions exist in which mucociliary
`clearance does not function properly. Primary ciliary dyskine-
`sia syndrome, a group of congenital disorders also known as
`Kartagener’s syndrome, is characterized by functional abnor-
`malities of the cilia and subsequent impairment of the normal cil-
`iary motility patterns in the respiratory and genitourinary tracts.
`The dysfunctional cilia within the respiratory tract are linked
`to increased occurrence of bronchiostatis, bronchial infection,
`and chronic rhinitis. If the application of drugs or formulation
`additives to the nasal mucosa results in similar patterns of dys-
`function, it is likely that similar clinical pathologies may oc-
`cur in chronic users of these medications (Donovan and Zhou
`1995). Patients with cystic  brosis also have impaired mucocil-
`iary clearance system, although their cilia are normal and func-
`tion well (Middleton, Geddes, and Alto 1993). The mucus of
`cystic  brosis patients has reduced water content, and the trans-
`port of this mucus has been observed to be delayed in vitro (Liote
`et al. 1989).
`In case of viral and bacterial infections, the mucociliary clear-
`ance system is compromised, most likely due to a loss of cilia
`but possibly also to a change in the rheological properties of
`the mucus (Lindberg 1994). Hospitalized patients in intensive
`care units often have impaired mucociliary transport, which is
`associated with the development of pneumonia and retention
`of secretion (Konrad et al. 1994). In diabetes mellitus patients,
`who are susceptible to nasal infectious diseases, nasal mucocil-
`iary clearance time was found to be signi cantly larger than in
`a group of nondiabetic controls (Sachdeva et al. 1993).
`To study mucociliary clearance, it is important to understand
`the pathology of the cilia. Cilia are motile hair-like appendages
`extending from the surface of epithelial cells. They beat in syn-
`chronized fashion in a highly complicated manner. The ciliary
`beat frequency (CBF) is regulated by several factors: tempera-
`ture, intracellular calcium ion, cAMP levels, and by extracellular
`ATP. The CBF of human nasal cells in vitro increases with in-
`±
`±
`±
`creasing temperature, between 5
`and 20
`C. Between 20
`and
`±
`45
`C, the frequency stabilizes at approximately 8–11 Hz and
`±
`±
`about 14 Hz between 32
`and 37
`C (Clary-Meinsez et al. 1992).
`The temperature dependency of cilia is mostly regulated by its
`
`axonemal enzymatic components, while the ciliary membrane
`has little effect. Extracellular ATP can increase the intracellu-
`lar calcium level in cell cultures, resulting in an increased CBF.
`The ciliary beat frequency is also increased by increasing the
`levels of intracellular cAMP and cGMP (Lansley, Sanderson,
`and Dirksen 1992; Green et al. 1995).
`As indicated, the function of mucociliary clearance is to pro-
`tect the nose and the lower airways from damage by inhaled
`noxious substances; therefore, impairment of this system is po-
`tentially harmful. The ef ciency of the mucociliary clearance
`system depends on the physiological control of CBF and on the
`rheological properties of the mucus blanket. Normal mucocil-
`iary transit time in humans is from 12 to 15 min. Transit times
`of more than 30 min are abnormal and are indicative of im-
`paired mucociliary clearance. Thus, average rate of nasal clear-
`ance is about 8 mm/min, ranging from less than 1 to more than
`20 mm/min (Andersen and Bende 1984).
`
`Mechanical Devices to Evaluate Mucociliary Clearance
`Many devices routinely measure CBF both in vivo and in
`vitro. High-speed cinematography estimates the frequency of
`ciliary waves; a video camera records the scene at high speed
`and afterward projects it at low speed for analysis. The time re-
`quired for the camera to provoke a cessation of movement is es-
`timated to assess ciliary activity (Gallay 1960; Sisson, Yonkers,
`and Waldman 1995; Gilain et al. 1993). With this technique, the
`cilia are illuminated with stroboscopic light  ashing at variable
`cycles per second, which when equal to the frequency of the cil-
`iary movement are perceived to the stationary (Andersen 1971).
`A photo multiplier transforms the light variations that result
`from mucociliary waves into voltage variations. After suitable
`ampli cation, the frequency is assessed (Dalhamn and Rylander
`1962). In a second technique, the cilia from the rabbit oviduct
`are illuminated with a laser beam. The spectrum of the scattered
`light, when analyzed, provides information about the frequency
`of the ciliary movement (Lee and Verdugo 1976, 1977). A photo-
`electric registration device measures tracheal CBF. Light trans-
`mitted through the cilia is detected by a phototransistor mounted
`in a microscope, which measures the frequency instantaneously,
`displaying the waveform on an oscilloscope connected to a tran-
`sient recorder (van de Donk, Zuidema, and Merkus 1980a). Pho-
`toelectronic detection is probably the most convenient means of
`quantifying CBF. It gives a complex spectrum of frequencies,
`but fast Fourier transform analysis of the analog signal gives
`a power spectrum of the  uctuating frequency. A disadvantage
`of using transmitted light is that only the beat frequency of the
`cilia at the edges of a piece of tissue explant can be measured.
`However, in cell cultures, it is possible to transmit light through
`the cell monolayer (Eshel, Crossman, and Priel 1985).
`
`Different In Vivo/In Vitro Methods to Measure
`Mucociliary Clearance
`±
`Usually CBF is measured at body temperature (37
`C), while
`the physiological range of nasal mucosa temperatures lies
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`230
`
`±
`
`M. QUADIR ET AL.
`±
`±
`±
`C, the
`and 40
`C. However, between 32
`and 35
`between 31
`nasal ciliary beat frequency was found to be independent of
`temperature. Optimal ciliary beat frequency was observed be-
`tween pH values of 7 and 10. pH values lower than 6 and higher
`or equal to 11 resulted in larger decreases in the ciliary beat
`frequency of chicken embryo trachea. The activity of the cil-
`iary beat is best evaluated in an isotonic solution (van de Donk
`et al. 1980a). The human amputated interior turbinate model
`was used to investigate the effect of chitosan on mucociliary
`transport rate (Aspden et al. 1997; Mason et al. 1995). Human
`turbinate begin to deteriorate approximately 4 hr postamputa-
`tion; thus, to ensure that turbinate mucus exhaustion would not
`in uence the results, all experiments were completed within 3 hr.
`After 15 min of chitosan contact, graphite particles were sprin-
`kled over the surface of the turbinates and their movement was
`recorded. Human turbinates are in limited supply, and the tissue
`is more fragile and sensitive to suboptimal conditions than the
`other methods. However, this human model was found to give
`reproducible results, which alludes to the possibility of identi-
`fying differences between the effects of various compounds on
`mucocilliary clearance rate and could lead to greater accuracy
`in predicting the effects of nasally applied substances in clinical
`situations.
`Clearance time also has been assessed by a standard saccha-
`rin taste test (Outzen and Svane-Knudesen 1993). This test is
`noninvasive and involves administering a saccharin formulation
`to one nostril and recording the time to taste the sweetness of
`the saccharin. The advantages of the saccharin clearance test as
`an indicator of mucocilliary clearance rate include its simplicity
`and relative inexpensiveness, which make it a routinely used and
`popular procedure in rhinology clinics.
`Techniques involving excised frog palates devices and em-
`bryonic chick tracheal tissues (Dalhamn 1955) also have been
`used to evaluate the ciliary movement. These methods are too
`expensive for routine testing and sometimes problematic to test
`on humans or animal models. However, these techniques are
`quantitatively quite accurate and reproducible but require spe-
`cialized equipment for data acquisition and analysis. In addition,
`excised tissues remain viable for a  nite period of time, thus re-
`stricting these techniques to the evaluation of acute drug-induced
`or short-term effects. There are several advantages of the ex vivo
`methods described above for assessing ciliotoxicity, including
`the opportunity for simulating therapeutic dosage regimens un-
`der conditions in which the animal’s natural defense mechanisms
`including mucus production and mucociliary clearance remain
`uncompromised.
`Donovan and Zhou (1995, 1996) developed a nonsurgically
`modi ed rat method to measure the clearance of nonabsorbable
`particles from the nasal cavity. Similar to other in vivo testing
`procedures (the saccharin test), this method measures clearance
`by collecting the marker as it enters the oral cavity following its
`transit through the nasal cavity. The clearance pattern was inves-
`tigated by measuring various kinetic parameters. The clearance
`of these particles from the nasal cavity follows simple  rst order
`
`kinetics. Both the rate (k or t90) and extent (AUC) of particle re-
`covery in these cases can be used to quantitatively compare drug
`induced changes in clearance. When particle clearance does not
`follow a de ned elimination order, the rate constant cannot be
`used for comparisons, but t90 and AUC values may be used for
`limited quantitative comparisons and additional qualitative as-
`sessments of changes in clearance patterns. These in vivo clear-
`ance studies also can be performed repeatedly thus enabling
`the time course of recovery of normal clearance patterns to be
`followed. Table 1 summarizes various types of approaches and
`methods used to evaluate the nasal toxicity of different drugs
`and excipients.
`Although cilliary motility has been observed by many in vivo
`and in vitro methods, little correlation exists between these meth-
`ods. Mucocilliary clearance rates are governed by interactions
`away mucus, cilia, and the intervening periciliary  uid. In vitro
`methods usually evaluate the effects of substances on individual
`components of clearance rates (i.e., CBF) and provide a good
`screening method for identifying substances with potential dele-
`terious effects on nasal mucosal structure and/or mucocilliary
`function. However, they cannot totally substitute for in vivo
`investigations. The effect of various substances on nasal mu-
`cocilliary clearance rates, CBF, and the nasal epithelium when
`applied in a clinical setting cannot be predicted from in vitro ap-
`proaches because factors such as dilution by mucus and limited
`contact with the mucosa cannot be accurately reproduced.
`
`Histological Studies
`Toxicological models were developed to compare the relative
`effects of different formulations on the morphology of the nasal
`mucosa. Scanning electron microscopy, used routinely to char-
`acterize the normal ultrastructure of the nasal respiratory epithe-
`lium, has proved an excellent technique for evaluating both gross
`structural alteration and speci c cellular changes induced by ex-
`posure to different chemicals (Ennis, Borden, and Lee 1990).
`The main limitation of this type of technique is model design,
`with exposure conditions in test models often more severe than
`what is encountered in a clinical situation. Thus, the recorded
`histological alteration may not represent those observed after
`single or chronic dosing in the clinic. Furthermore, these mod-
`els give a comparative qualitative evaluation that necessitates
`the use of multiple individuals with histopathological expert to
`evaluate the results.
`
`Release or Alteration of Nasal Chemicals
`Release of marker compounds from the nasal cavity also may
`evaluate potential damage to the nasal epithelium (Emmeline
`et al. 1995). Histological studies have disclosed that acid phos-
`phatase activity is present in the squamous and respiratory ep-
`ithelium. This activity is highest in the sensory cells of the ol-
`factory epithelium, whereas the mucus and cilia do not contain
`acid phosphatase activity. The release of acid phosphatase is
`therefore an indication of nasal epithelial damage, especially in
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`TOXICOLOGICAL IMPLICATIONS OF NASAL FORMULATIONS
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`231
`
`TABLE 1
`Methods of evaluating nasal formulations
`
`Method
`
`In vivo/in vitro
`
`Specimens
`
`References
`
`Clearance
`Video camera
`
`Photoelectric
`
`Laser beam
`Cell culture
`Interior turbinate
`Saccharine taste test
`Frog palate mode
`
`Others
`Kinetic parameters
`Morphology
`Release of marker
`Electrical membrane
`resistance
`Vasomotion effect
`Direct access to CNS
`Direct access to CNS
`
`In vitro
`In vitro
`In vitro
`In vitro
`In vitro
`In vitro
`In vitro
`In vitro
`In vivo
`In vitro
`In vitro
`In vitro
`
`In vivo
`In vitro
`In vivo
`In vitro
`
`In vivo
`In vivo
`In vivo
`
`Gallay 1960
`Guinea pig
`Sisson et al. 1995
`Human
`Gilian et al. 1993
`Human
`Dalhamn 1962
`Rabbit
`van de Donk et al. 1980a
`Chicken embryo
`Lee and Vardugo 1977
`Rabbit oviduct
`Eshel et al. 1985
`Frog
`Mason et al. 1995
`Human
`Outzen et al. 1993
`Human
`Chicken embryo Dalhman 1955
`Frog
`Batts et al. 1989
`Frog
`Gizurarson et al. 1990
`
`Rat
`Rat
`Rat
`Rabbit
`
`Rabbit
`Rat
`Rat
`
`Donovan et al. 1995
`Lee et al. 1995
`Emmeline et al. 1995
`Hosoya et al. 1994
`
`Bende et al. 1992
`Chou and Donovan 1996
`Sakane et al. 1997
`
`the olfactory region. Cholesterol also may be released in the
`nasal  uid as a result of the interaction of absorption enhancers
`with the nasal epithelium (Emmeline et al. 1995). The extent
`of release of total protein and the enzymes, such as lactate de-
`hydrogenase (LDH) and 5
`-nucleotidase (5
`-ND), correlate with
`the extent of damage sustained by the nasal mucosa.
`Membrane-bound 5
`-nucleotidase release into the nasal per-
`fusate gives an indication of the level of membrane perturbation,
`while release of LDH, a cystolic enzyme indicates the amount
`of cell leaching or lysis. The total protein release data, although
`not very speci c as to the type of damage, provide a general
`indication of the extent of irritation (Shao and Mitra 1992a). Un-
`fortunately, the analytical methods used to determine the levels
`of release of these marker compounds often are not highly sen-
`sitive and thus may complicate interpretation of the results. In
`addition, many excipients used in nasal formulation may inhibit
`the enzyme activity. Therefore, although the damage occurs, the
`enzyme activities may not be detected.
`
`Membrane Resistance Measurement
`Recently, the measurement of the electrical membrane resis-
`tance across the nasal mucosa has been used as a criterion to
`evaluate the damage caused by enhancers (Hosoya et al. 1994).
`Hosoya evaluated electrical membrane resistance (Rm) after ap-
`plying absorption enhancers using the Ussing chamber tech-
`
`nique. Although Rm values were kept constant in the absence
`of an enhancer, the value decreases drastically after applica-
`tion of enhancer. The magnitude of Rm change correlated well
`with the morphological changes shown by scanning electron
`microscope. It was postulated that a signi cant decrease in Rm
`resulted when an enhancer opened tight junctions or made new
`pore routes. But the behavior of Rm change with variation in
`exposure time and concentration of enhancer was not well doc-
`umented. In this study, nasal mucosa were stripped from the
`nasal septum and placed in standard Ringer’s solution. These
`excised tissues remain viable for a  nite period of time, thus
`restricting the evaluation to short-term effects.
`
`Vascular Reactions
`The high vascularity and large surface area of the nasal mu-
`cosa make it a suitable site for rapid absorption of drug through
`the nasal cavity. Blood  ow and therefore drug absorption may
`often depend upon the vasodilation and vasoconstriction of the
`blood vessels. Various methods have been used for experiential
`evaluation of vascular reactions in the nasal mucosa. Oxymeta-
`zoline has been shown to affect the vascular permeability of the
`nasal mucosa as a result of vasoconstriction (decrease in blood
` ow) and/or change in the permeability characteristics (Bende
`et al. 1992). In humans, changes in the blood content of the nasal
`mucosa have been studied indirectly using techniques such as
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`M. QUADIR ET AL.
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`rhinomanometry and plethysmography (Ralston and Kerr 1945).
`Nasal blood  ow also has been estimated indirectly by measur-
`ing changes in temperature or thermal conductivity in the mu-
`cosa (Bende and Flisberg 1985). Photometrically determined
`color changes within the nasal mucosa also have been used to
`indicate changes in blood  ow (Jackson and Martinez 1965). A
`variety of drugs have been shown to affect the blood  ow within
`the nasal mucosa. For example, clonidine decreases the blood
` ow (Anderson and Bende 1984) whereas histamine, albuterol,
`isoproterenol, and phenylephrine were shown to increase blood
` ow (Lung and Wang 1985; McLean et al. 1976).
`
`EFFECT OF ACTIVE DRUGS, EXCIPIENTS, AND
`FORMULATION VARIABLES
`During the development process, the complete formulation
`must be evaluated for toxicological response following chronic
`dosing as well as for less damaging sensitivity reactions, since
`these responses may be due to the active ingredients or the ex-
`cipients used in the nasal formulation. Absorption enhancers are
`often necessary to increase the bioavailability of drugs. Often
`large molecules, such as peptides and proteins, require enhancers
`to reach an effective level of absorption from the nasal mucosa.
`Preservatives also are used for multidose formulations. Certain
`formulation variables like pH, ionic strength, osmolarity, charge,
`and concentration of buffer species also may contribute to the
`toxic response of the formulation. Thus, evaluation of the overall
`effect of active drug, excipients, or formulation variables aids
`the pharmaceutical scientist in formulating a product that does
`not irritate the nasal mucosa and that in turn may provide more
`clinically acceptable nasal formulations for patients with chronic
`use.
`
`Effects of Active Drugs
`A variety of drugs have been shown to be well absorbed by
`the nasal mucosa with resultant bioavailability comparable to
`intravenous or subcutaneous administration. But the nasal mu-
`cosa does not have the same tolerability to all drugs. Most of the
`antimicrobial agents that have been studied demonstrated a nom-
`inal effect on the ciliary beat frequency. Yet benzyl penicillins
`(10,000 U/ml) and ampicillin (1%) exhibited little ciliotoxic-
`ity in vitro. The ciliotoxic effects of sulfonamides were more
`pronounced than those of the penicillins when administered in
`a similar therapeutic range but were still reversible. Neomycin
`(0.35%) and chloramphenicol (0.4%) inhibited protein synthe-
`sis, though only chloramphenicol was found to penetrate the
`cells and inhibit protein synthesis in eukaryotic cells (van de
`Donk et al. 1982a). Bacitracin, in an in vitro study when diluted
`to ten times less than the minimum therapeutic level, depressed
`ciliary activity dramatically and irreversibly. In contrast, in vivo
`studies showed that even though there was a decrease in the
`nasal clearance rate by bacitracin at the same concentration as
`used in the previously described in vitro study, normal clear-
`ance resumed within 48 hr (Donovan and Zhou 1995). The mor-
`
`phology of excised nasal mucosa exposed to bacitracin showed
`signi cant changes in ciliary organization after 30 min and was
`completely denuded of cilia after 120 min.
`During the past decade, extensive research has been per-
`formed on the potential importance of the delivery of peptides
`and proteins via the nasal route. Most of this research was de-
`signed to evaluate the bioavailability of these macromolecules
`when given alone or with surfactants and/or various enhancers.
`Peptides and proteins usually do not have a signi cant deleteri-
`ous effect on the nose. For example, good tolerability was found
`for somatostatin in animals (Ainge et al. 1994) and in the hu-
`man nasal mucosa (Harris et al. 1992; Weeke et al. 1992). No
`damage to the ciliated cells and no signi cant increase in mu-
`cus production were observed after administration of somato-
`statin (Fraissinette et al. 1995). Insulin did not cause cilio-inhi-
`bition in vitro in rats and only resulted in a slight, reversible
`decrease in the mucocilliary transport rate in frog palate mode
`(Gizurarson 1990). Salmon calcitonin had no effect on mucocil-
`liary transport in the frog palate model nor on the ciliary beat
`frequency of mouse septal membranes grown in cell cultures
`(Honda et al. 1992).
`Additional study showed human insulin, salmon calcitonin,
`glypressin, and desmopressin did not have any signi cant dele-
`terious effect on nasal mucosa (Bende et al. 1986; Critchely
`et al. 1994). In vitro studies showed that most local anesthet-
`ics severely but reversibly affected ciliary beat frequency. The
`reversibility diminished in the order lignocaine, cocaine to bu-
`tacaine. However, the effect at pH 6 was less compared with
`that at pH 7 (van de Donk et al. 1982b). This may be due to
`the pka values of this group of drugs, which are from 8 to 10.
`At a lower pH, the undissociated portion of the drug that dif-
`fuses through the cell membrane is small compared with the
`protonated fraction thus reducing effectiveness at lower pH. In
`an in vitro study, cocaine was found to decrease ciliary beat
`frequency at a concentration of 1.75% or higher in human nasal
`cilia. At a 7% concentration, ciliary inhibition was only partially
`reversible (Ingels and Nijziel 1994). Interestingly, in an in vivo
`chicken model, a 5% cocaine solution produced an increase of
`mucociliary transport, while a 20% cocaine solution caused a
`complete ciliostasis effect (Ukai, Sakakura, and Saida 1985).
`In vivo studies showed lidocaine HCl signi cantly decreased
`clearance rate. While in vitro results indicated that although li-
`docaine rapidly decreased ciliary beat frequency, the beating fre-
`quency slowly recovered after this drug solution was rinsed from
`the tissue section with Locke-Ringer’s solution (van de Donk
`et al. 1982b). Corssen (1973) reported that 0.01% lidocaine
`caused no change; 0.1% slightly increased, and 5–20% signi -
`cantly decreas