Nasal drug delivery: A direct approach to the cerebrospinal fluid? Berg, M. van den

(1)

Nasal drug delivery: A direct approach to the cerebrospinal fluid?

Berg, M. van den

Citation

Berg, M. van den. (2005, April 14). Nasal drug delivery: A direct approach to the

cerebrospinal fluid?. Retrieved from https://hdl.handle.net/1887/1999

Version: Corrected Publisher’s Version

(2)

Nasal drug delivery:

A direct approach to the cerebrospinal fluid?

Proefschrift

ter verkrijging van

de graad Doctor

aan de Universiteit Leiden,

op gezag van de Rector Magnificus Dr. D.D. Breimer,

hoogleraar in de faculteit der Wiskunde en

Natuurwetenschappen en die der Geneeskunde,

volgens besluit van het College voor Promoties

te verdedigen op donderdag 14 april 2005

te klokke 15.15 uur

door

(3)

Promotiecommissie

Promotor: Prof. dr. F.W.H.M. Merkus

Copromotor: Dr. J.C. Verhoef

Referent: Dr. E. Björk (Uppsala, Sweden)

Overige leden: Prof. dr. G.J. Mulder Prof. dr. J.A. Bouwstra Prof. dr. H.J. Guchelaar Prof. dr. M. Danhof Prof. dr. E.R. de Kloet

(4)

The investigations described in this thesis were performed at the subdivision of Pharmaceutical Technology and Biopharmaceutics of the Leiden/ Amsterdam Center for Drug Research, Leiden University, The Netherlands.

(5)

(6)

1

(9)

1. Introduction

The targeting of drugs to the brain is a major problem in drug delivery research. The tight construction of the brain capillary endothelial cells that form the blood-brain barrier (BBB), is the main obstacle that needs to be overcome before drugs can enter the central nervous system (CNS). Only small lipophilic compounds (M_w < 600 Da) and nutrients and peptides which have a carrier-mediated transport system in the BBB can be transported from the systemic circulation into the brain (108, 132). To enhance the uptake into the CNS of drugs that do not meet these crite-ria, several strategies have been investigated to manipulate the barrier function of the brain capillary endothelial cells. However, there is still no satisfactory method to target drugs across the BBB.

(10)

2. The nose

The inhaled air is warmed and humidified in the nasal cavities. The mu-cus filters the air by clearing dust particles, bacteria and other inhaled substances from the nasal cavity by the mucociliary clearance. Other na-sal functions are vocalisation and olfaction.

The nasal cavity is split into two symmetrical halves by the nasal septum. The structure of each cavity is further determined by the shape of three turbinates: the inferior, middle and superior one. The two halves are sub-divided in the vestibule, lined with skin-like epithelium, the respiratory and the olfactory area (Figure 1).

2.1. The respiratory and olfactory epithelium

The respiratory epithelium, covering the main part of the nasal cavity (~ 160 cm2_{in humans (142)), consists of four main cell types: ciliated and} non-ciliated columnar cells, goblet cells and basal cells (Figure 2). The epithelial cell layer is covered with mucus, which is produced by the gob-let cells and cleared by the beating of the cilia, the so-called mucociliary clearance. This clearance mechanism protects the respiratory tract in-cluding the lungs from bacteria and other harmful exogenous compounds. The respiratory mucus layer contains several isoforms of the metabolis-ing enzyme family P-450. The olfactory mucus layer has even a higher capability to degrade xenobiotics than the respiratory one. However, be-cause of the high vascularisation of the latter mucus layer nasally admin-istered drugs are quickly absorbed and hardly metabolised by the zymes. Secondly, the small amount of specific cytochrome P-450 en-zymes decreases the risk on degradation compared to other P-450 con-taining organs like the liver (131).

The olfactory epithelium is located at the top of the nose between the superior turbinate and the roof of the nasal cavity and covers only 10 – 20 cm2_{in humans. This is about 8 % of the total nasal surface area,} whereas in rats the olfactory area comprises 50 % of the nasal cavity (41). The olfactory epithelium is build up by three main cells. The olfactory receptor neurons (ORN) are bipolar cells connecting the olfactory bulb with the nasal cavity. The axons of the ORNs extend from the cell body in the olfactory epithelium through the cribriform plate into the

(11)

Figure 1 Anatomic representation of the human nasal cavity; a olfactory area and super conchae; b respiratory area comprising the middle (c) and inferior (d) conchae; e vestibular region; f palate.

(12)

tory bulb and synaps with juxtaglomerular neurons and with tufted and mitral cells in the glomeruli (139). The other end of the ORN, a dendritic knob containing several dendritic cilia, terminates in the olfactory epi-thelium (Figure 3). The ORNs are located in between the supporting or sustentacular cells. These are large columnar cells covered with microvilli at the apical side (103). The functions of these cells are unclear, but prob-ably include the secretion of mucus, insulation of ORNs and guidance of developing basal cells (103). It is also suggested that the supporting cells are a source of xenobiotic-metabolising enzymes (68). Adjacent to the basal lamina of the olfactory epithelium a layer of basal cells is lo-cated, which is covered with a second layer of these cells (104). The latter one contains the so-called globose basal cells, which undergo continuous mitotic activity and replace ORNs under normal conditions and follow-ing injury (103). Other structures located in the olfactory mucosa are Bowman’s glands and blood and lymphatic vessels (99).

2.2. Nasal transport routes

After nasal delivery, drugs will at first reach the respiratory epithelium, from where compounds can be absorbed into the systemic circulation. When a nasal drug formulation is delivered deep and high enough into the nasal cavity, the olfactory area may be reached and drug transport via the ORNs might occur. Two possible routes exist by which molecules can be transported from the olfactory epithelium into the brain and/or CSF. First the epithelial pathway, considered as a fast route (passage takes < 2 hours (10, 58)), where compounds pass paracellularly across the olfac-tory epithelium into the perineural spaces, crossing the cribriform plate and ending up in the subarachnoid space filled with CSF. Then the mol-ecules can diffuse into the brain tissue or will be cleared by the CSF flow into the lymphatic vessels and subsequently into the systemic circulation. The second possibility is the olfactory nerve pathway, which is a very slow route (passage takes > 1 day (10, 18, 138)). Compounds may be internal-ised into the ORN and pass inside the neuron through the cribriform plate into the olfactory bulb. From here further transport into the brain can occur by bridging the synapses between the neurons. Once a drug is in the brain, it can be further influenced by BBB efflux transporter sys-tems like P-glycoprotein (67, 134). All possible transport routes after na-sal drug administration are depicted in Figure 3 and 4.

(13)

Figure 3 Schematic presentation of the olfactory mucosa; a olfactory receptor neuron; b supporting cell; c basal cell; d Bowman’s gland; e lymphatic capilaries; f fila olfactoria; g Schwann’s cells; h perineural cells; i dura mater; j arachnoid mater; A olfactory mucosa; B lamina propria; C cribriform plate; D subarachnoid space; E olfactory bulb.

(14)

3. The neuronal connection

The ORN’s connect the nasal cavity with the brain and the CSF. For years it is known that substances injected into the CSF or brain tissue are transported into the nasal mucosa and lymphatic vessels (15, 30). This is in agreement with the physiological CSF drain, which starts at the choroid plexi where it is produced. From the ventricles inside the brain the CSF flows towards the spinal cord and back into the subarachnoid space sur-rounding the outer brain surface. Subsequently it is drained into the su-perior sagittal sinus across the arachnoid granulations. The CSF is drained away from the CNS by movement into blood vessels within the sagittal sinus and by flow into the deep cervical lymphatics (15). The drainage of dyes and micro-organisms from the CSF and/or brain to the lymphatics and nasal mucosa has already been reviewed by Yoffey (159) and Jackson et al. (80), although the exact mechanism was still unknown to these au-thors. This mechanism was further elucidated by Erlich et al. (49), who injected ferritin into the lateral ventricle in rabbits and found that this substance was transported by the CSF drain along the olfactory pathway into the nasal mucosa. Microscopic studies showed that ferritin was trans-ported from the ventricles into the nasal mucosa via an open pathway between the subarachnoid space and the olfactory epithelium.

By nasal application of several dyes, the transport route in the opposite direction was demonstrated (6, 53, 154). This observation was supported by experiments with viruses and metals (98, 112, 146, 149). However, the entrance of viruses and metals may cause toxic effects in the CNS, which have to be avoided (17, 63, 70, 72, 97, 160).

Some studies describing the nose-CSF/brain transport of dyes, viruses and metals show that these compounds are directly transported from the nasal cavity into the CSF and/or brain. However, for several other sub-stances like small molecular weight drugs, peptides and high molecular weight compounds there is not a consistent answer to this transport route. As mentioned in the Introduction, the used experimental set-up and the methodology of data analysis can influence the interpretation of the sults obtained. Therefore, the different methodologies used in this re-search area will be discussed below.

(15)

4. How to study nose-to-CSF/brain drug transport?

First, the question of possible direct drug transport from the nasal cavity to the brain/CSF will be discussed by a theoretical approach keeping in mind the possible transport routes of a drug after nasal delivery as de-picted in Figure 4. Subsequently, the experimental models published in the literature will be described.

4.1. Theoretical study design

(16)

plasma CSF AUC AUC ratio plasma CSF/ = _{Equation 1}

The CSF/plasma ratio of a compound is expressed by the area under the CSF concentration-time curve (AUC_CSF) divided by the area under the plasma concentration-time curve (AUC_plasma) following the same admin-istration route (75, 153). The AUC value of a drug, determined in plasma or CSF, is a quantitative measure that reflects the complete uptake profile of the compound in the specified biocompartment. Therefore, it is pre-ferred to relate AUC values rather than drug concentrations at specific time points because such time points give limited information on the total uptake profile. Instead of AUC_CSF, it is also possible to investigate AUC_brainvalues. However, since the AUC values are more often calcu-lated for CSF drug concentrations than for brain drug concentrations, the term AUC_CSF is used in this Thesis.

In case of direct drug transport from the nasal cavity, it is expected that the CSF/plasma ratio following intranasal delivery will be significantly higher than that after intravenous administration. When this ratio is equal or lower than the intravenous one, the observed drug transport can be considered as systemically and not via the ORNs.

Besides a pharmacokinetic analysis, nose-brain/CSF transport can also be investigated on the level of pharmacodynamics (56). However, it is still necessary to monitor plasma levels when studying drug effects, be-cause a larger change in pharmacodynamic effect can be due to higher drug plasma levels and not necessarily to a direct drug uptake from the nasal cavity into the CNS. Because it is difficult to relate a pharmacody-namic effect to plasma levels following different administration routes, the drug plasma profile after nasal delivery should be mimicked by intra-venous administration of the same drug. In this way it is possible to compare drug effects after nasal and intravenous administration without relating them to the obtained plasma levels (119). When investigating pharmacodynamics, a control treatment is generally included, as the change in effect is the actual point of measurement.

(17)

In conclusion: Using the pharmacokinetic approach the nasal route of drug administration should be compared with intravenous delivery, if possible resulting in similar plasma levels as following nasal administra-tion. The drug distribution over CSF and plasma or brain tissue and plasma can be expressed in terms of CSF/plasma ratios based on the obtained AUCs. Only when the CSF/plasma ratio after nasal deliverysignificantly exceeds the one after intravenous delivery, the drug transport route can be considered as directly from the nasal cavity into the CSF and/or brain tissue. In pharmacodynamic studies it is necessary to achieve similar drug plasma levels after intranasal and intravenous administration, in order to compare the pharmacological effect after different administration routes. 4.2. Study designs used in the literature

The investigation of drug transport from the nasal cavity into the CSF or brain requires animal experiments. Rats and mice are used most often in such studies, although the anatomy of the nasal cavity of rodents is dif-ferent from that in humans. Rats are widely used in nasal drug absorption studies (77) and therefore it is obvious that this species is frequently used for nose-brain transport studies as well. The advantage of mice is that

Table I References of the used publications per research category

(18)

large numbers of animals can be used easily, which is necessary when sampling brain tissue. The most striking anatomical difference between the human and rat nose is the distribution of the respiratory and olfac-tory epithelium over the nasal cavity as mentioned above in paragraph 2.1. The literature about nose-to-brain/CSF drug transport can be di-vided into four categories: nose-brain, nose-CSF, nose-brain and CSF, and pharmacodynamic (PD) research. These categories will be further discussed with respect to the study designs that are used in this research field.

4.2.1. Nose-brain research

In 64 publications (Table I) of the investigated literature (134 papers), brain tissue was sampled for examining the nasal pathway to reach the CNS. The most commonly used species are rats and mice, but only a few studies are known using rabbits (29, 53, 106, 111), pigs (87, 89, 143), monkeys (10, 13, 51, 64), dogs (154) and hamsters (100). Only a few of these studies compared nasal with intravenous drug administration. In most papers intranasal administration was compared with a control ad-ministration like placebo, vehicle or no treatment, and a delivery route other than the intravenous one.

In the majority of the nose-brain studies, brain tissue is sampled only, without monitoring the drug plasma levels. Generally, the brain tissue is sampled by dissection or by slicing the brain tissues. The dissected tis-sues are analysed by determining radioactivity or, after extracting the drug of investigation, by HPLC or radioimmunoassay. The brain slices are analysed either by autoradiography or by staining techniques using microscopy. Investigating the drug transport via the ORNs using microscopy still needs comparison with intravenous injection, as intrave-nously administered substances can reach the ORNs via transport from the blood into the CSF, which fills the perineural spaces between the ORNs (16, 80).

The majority of the studies using brain tissue sampling investigated the possibility of direct nose-brain transport in a qualitative way using auto-radiography or microscopic analysis. There are only 3 publications show-ing quantitative data on nose to brain transport (27, 28, 76). Chow et al.

(19)

(28) calculated the brain/plasma ratio for three different brain areas fol-lowing intranasal administration of cocaine, and did not found any direct transport. This is supported by Hussain et al. (76) in their study with a cognition enhancer. The observed brain/plasma ratios after intranasal and intravenous delivery appeared to be similar. In another report by Chow et al. (27) the brain/plasma ratio was higher after intranasal admin-istration of benzoylecgonine compared to that after intravenous injec-tion.

The reason for the very few number of kinetic studies sampling brain tissue, is the large number of required animals, which makes it also labo-rious and expensive. When sampling brain tissue only, one sample per animal per time point has to be taken. This also increases the variability in the obtained data.

In summary, microscopic analysis of brain tissue gives a qualitative an-swer whether or not there is direct drug transport. A disadvantage is that microscopy does not give an indication about the proportion of the ad-ministered drug that is transported from the nasal cavity into the brain. Direct transport may be concluded after spotting only a few molecules in or along the olfactory tract. However, this is probably not satisfactory for achieving pharmacologically relevant drug concentrations in the brain. 4.2.2. Nose-CSF research

(20)

mal can be collected. This increases the number of animals needed to obtain a complete drug uptake profile in CSF and/or limits the data on drug uptake in CSF. Subsequently, the variability between data will in-crease compared to the results obtained from one animal. Secondly, there is a high risk on contamination of the CSF with blood. This can be the reason of measuring enhanced drug levels in CSF, as blood contaminat-ing the CSF will contribute considerably to these drug concentrations. Several papers reported that CSF sampling was stopped as soon as blood appeared and that this last fraction was discarded. However, this can still be too late to stop the CSF collection because it may already be contami-nated with blood, even if not visible with the eye.

Only a few papers describe serial CSF sampling in rats in this field of research (7, 23, 25). Chou and Donovan investigated the CSF penetra-tion of intranasally administered antihistamines (23) and local anaesthet-ics (25) using serial CSF sampling by cisternal puncture, but with replace-ment of CSF via a needle positioned in the lateral ventricle. The advan-tage of this CSF sampling method is that CSF and blood samples can be collected serially from one animal. The collected CSF samples are small (about 30 µl), which may be disadvantageous for drug analysis, but ex-cludes possible blood contamination. Bagger and Bechgaard (7) devel-oped a microdialysis method to sample extracellular fluid (ECF) in the left and right striatum in rats. The sampled ECF is replaced with artificial CSF and sampling from the second half of the brain serves as a negative control in case of direct nose-brain drug transport. Another advantage of the model is the ability to insert the microdialysis probe in the brain area of interest in order to monitor the drug concentration at the site where the pharmacological effect will take place. A disadvantage of this model and the previous one is the invasive character and the risk on blockage of the implanted cannula and probe, which makes it difficult to use the rats for both intranasal and intravenous treatment. This would allow using the rats as their own control and therefore reducing the number of animals needed.

In about half of the nose-CSF transport studies the drug transport route from the nasal cavity to the CSF compartment is not determined by com-paring CSF/plasma ratios after nasal and intravenous delivery.

(21)

In some reports the CSF/plasma ratio is calculated for concentrations at particular time points. (4, 127, 128, 133, 134, 158). Other reports based their conclusion of direct nose-CSF transport only on increased drug uptake into CSF after nasal drug delivery compared to intravenous ad-ministration at one time point only, regardless from the observed plasma levels (3, 14, 31, 35, 36, 126). The information given by these concentra-tion ratios is not satisfactory to draw clear-cut conclusions on the uptake route of the drug after nasal delivery, but can only serve as an indication. Taking together, studying nose-CSF drug transport requires drug absorp-tion and uptake profiles in plasma and CSF, respectively, after both intra-nasal and intravenous delivery. A serious drawback in this type of experi-ments is the serial collection of CSF, which in small laboratory animals like rats is difficult to perform due to their small CSF volume (about 300 µl (37)). In humans the sampling of CSF is also rather difficult because of the invasive character. In healthy volunteers CSF was sampled by us-ing an interspinal catheter (14). Merkus et al. (101) obtained CSF from neurosurgery patients provided with a CSF drain necessary for their treat-ment. If it is not possible to collect CSF in a serial way because of meth-odological reasons, the results obtained should be used as an indication only.

4.2.3. Nose-brain and CSF research

Only 7 publications (Table I) of the selected literature describe experi-ments investigating plasma, CSF and brain tissue following nasal drug delivery. Four of these studies were carried out in rats (22, 24, 35, 129), whereas monkey (54), rabbit (130) and mice studies (97) were noticed only once. Also in these studies intravenous drug administration is the reference treatment used most often.

(22)

In the rat study by Chou and Donovan (24) CSF and brain tissue AUC values after nasal and intra-arterial delivery of lidocaine in rats were com-pared. The reported nasal/intra-arterial AUC ratios suggest direct trans-port to the CSF when sampling by cisternal puncture, but not when us-ing microdialysis as samplus-ing technique at different brain areas. Never-theless, these results are difficult to interpret, because nasal plasma ab-sorption data of lidocaine are not available.

In another rat study, the direct transport of tritiated dopamine was claimed (35). Comparable blood levels were found after nasal and intravenous administration. However, in blood, CSF and brain tissue total radioactiv-ity was measured, which does not distinguish between the intact com-pound and possibly formed metabolites.

The uptake of 5-fluorouracil into CSF and brain tissue was studied by Sakane et al. (129) following nasal instillation, nasal perfusion or intrave-nous administration in rats. After nasal perfusion enhanced drug trans-port to the cortex was found compared to intravenous administration. However, a volume of 8 ml was perfused through the rat’s nasal cavity for 30 min, which is a very aggressive method for nasal administration. Firstly, to prevent swallowing of a nasal formulation, no more than 20 µl per nostril should be applied to rats. Secondly, the residence time of the administered drug is not restricted by the mucociliary clearance, thereby prolonging the drug absorption phase. The distribution of 5-fluorour-acil over the cerebral cortex and plasma was expressed as the cortex/ plasma concentration ratios against time. This method of data analysis is remarkable, as it is possible to calculate the AUC_plasma and AUC_brain values for each delivery, making the interpretation of the results easier and more clear-cut.

In summary, to obtain both CSF and brain tissue samples requires a large number of experimental animals, but on the other hand it provides more information on the CNS distribution of the nasally administered drug. 4.2.4. Nasal drug delivery and pharmacodynamics

In the three previous sections the discussed experimental methods were focused on determining the drug concentration in CSF and/or brain

(23)

tissue and plasma following nasal administration. This section will de-scribe methods monitoring the pharmacodynamic effect of the adminis-tered drug as the main parameter. Humans are the most investigated spe-cies in studies describing the relation between intranasal drug delivery and the obtained CNS effects. This is explained by the fact that in hu-mans pharmacological effects can be monitored more easily than CSF drug concentrations. The majority determined CNS effects by monitor-ing the change in event-related brain potentials at one or more brain sites. One study investigating the nose-to-brain transport of the peptide drug angiotensin II measured the effects at peripheral sites: vasopressin and norepinephrine release was determined by measuring their plasma concentrations and the systemic vascular resistance was monitored (40). In animal studies, rats and mice are the most frequently used species, whereas monkeys were studied twice (52, 124) and dogs (20), rabbits (92) and pigs (105) only once. The effects studied in animals are mainly of neurochemical, histological or behavioural type of studies. For instance, the uptake of cocaine, L-DOPA methylester and amphetamine into the neostriatum in rats was determined by monitoring the neurochemical effects of these drugs on the dopamine, DOPAC and HVA levels in that brain area using microdialysis (38). Gozes et al. (65) studied the effect of a vasoactive intestinal peptide (VIP) analogue in rats on learning and memory behaviour. The development of neurons, plaques, astrocyte tox-icity, lesions, tissue degeneration and neuron staining are examples of histological effects monitored after nasal drug delivery. It is obvious that such effects are easier to monitor in laboratory animals than for example event-related brain potentials in humans.

In these pharmacodynamic investigations a control treatment seems to be more important than comparison of the intranasal and intravenous routes of drug administration. Other administrations used as reference in these pharmacodynamic investigations differ in nasal delivery condi-tions like dose, formulation composition or drug and therefore can hardly be used as control treatment.

(24)

monitored and in most of these papers no CSF levels. This means that the majority of these pharmacodynamic studies bases the drug transport route to the brain after nasal delivery on effect monitoring only, without taking into account the drug distribution over plasma and CSF or brain tissue. Considering the pharmacokinetically based CSF/plasma ratio as a key parameter necessary to determine whether or not the drug is directly transported from the nasal cavity into the CNS, gives these pharmacody-namic studies a weak basis for clear-cut conclusions.

From the discussion above it is evident that the majority of the studies in the area of nose-brain/CSF research do not meet the criteria as outlined in the theoretical study design (see 4.1.). Therefore, the results obtained in these studies will be evaluated in the General discussion and conclusions (Chapter 8) for their relevance and conclusions if direct drug transport from the nasal cavity to the CNS is justified or not.

5. Study objectives

The main objective of this Thesis was to investigate the relevance of drug transport from the nasal cavity directly into the CSF in rats. This was accomplished by fulfilling the following aims. Firstly, a rat model was developed in order to take serial CSF and blood samples from one ani-mal after nasal or intravenous drug administration. The intravenous de-livery device was set-up in order to simulate the drug plasma profile ob-tained after nasal administration to create similar experimental condi-tions for both delivery routes.

The developed rat model was subsequently used to examine the direct nose-to-CSF transport of the lipophilic compounds hydrocortisone, estradiol, progesterone and melatonin and the hydrophilic compounds hydroxocobalamin (M_w = 1.3 kDa) and fluorescein isothiocyanate-labelled dextran (M_w = 3.0 kDa). In addition, for the compound melatonin and the hydrophilic substance hydroxocobalamin the obtained results in rats were compared with those of two clinical studies in order to investigate the predictive value of the developed rat model for the situation in hu-mans.

(25)

6. References

1. Anand Kumar, T.C., David, G.F.X., Kumar, K., Umberkoman, B., and Krishnamoorthy, M.S. (1976) A new approach to fertility regulation by interfering with neuroendocrine pathways. In: Neuroendocrine Regulation of Fertility, Editor: Anand Kumar, T.C.; Karger, Basel, 314-322.

2. Anand Kumar, T.C., David, G.F.X., and Puri, V. (1979) Nasal spray and contraceptives. In: Recent Advances in Reproduction and Regulation of Fertility, Editor: Talwar, G.P.; Elsevier/North-Holland Biomedical Press, Amster-dam, 49-56.

3. Anand Kumar, T.C., David, G.F.X., Sankaranarayanan, A., Puri, V., and Sundram, K.R. (1982) Pharmacokinetics of progesterone after its admin-istration to ovariectomized rhesus monkeys by injection, infusion, or nasal spraying. Proceedings of the National Academy of Sciences of the United States of America 79 4185-4189.

4. Anand Kumar, T.C., David, G.F.X., Umberkoman, B., and Saini, K.D. (1974) Uptake of radioactivity by body fluids and tissues in rhesus monkeys after intravenous injection or intranasal spray of tritium-labelled oestradiol and progesterone. Current Science 43 435-439.

5. Ang, V.T.Y. and Jenkins, J.S. (1982) Blood-cerebrospinal fluid barrier to arginine-vasopressin, desmopressin and desglycinamide arginine-vaso-pressin in the dog. Journal of Endocrinology 93 319-325.

6. Arnold, W. and Vosteen, K.H. (1973) Zur sekretorischen Aktivitat der Interdentalzellen des Limbus Spiralis. Acta Otolaryngologica (Stockholm) 75 192-202.

7. Bagger, M. and Bechgaard, E. (2004) A microdialysis model to examine nasal drug delivery and olfactory absorption in rats using lidocaine hydro-chloride as a model drug. International Journal of Pharmaceutics 269 311-322. 8. Bagger, M.A. and Bechgaard, E. (2004) The potential of nasal application for delivery to the central brain-a microdialysis study of fluorescein in rats. European Journal of Pharmaceutical Sciences 21 235-242.

9. Baker, H. and Spencer, R.F. (1986) Transneuronal transport of peroxidase-conjugated wheat germ agglutinin (WGA-HRP) from the olfactory epi-thelium to the brain of the adult rat. Experimental Brain Research 63 461-473.

(26)

Av-system in mouse, rat, and squirrel monkey. Journal Of Comparative Neurology 251 260-280.

11. Barnett, E.M. and Perlman, S. (1993) The olfactory nerve and not the trigeminal nerve is the major site of CNS entry for mouse hepatitis virus, strain JHM. Virology 185-191.

12. Bergstrom, U., Franzen, A., Eriksson, C., Lindh, C., and Brittebo, E.B. (2002) Drug targeting to the brain: Transfer of picolinic acid along the olfactory pathways. Journal of Drug Targeting 10 469-478.

13. Bodian, D. and Howe, H.A. (1940) An experimental study of the role of neurones in the dissemination of poliomyelitis virus in the nervous sys-tem. Brain 63135-162.

14. Born, J., Lange, T., Kern, W., McGregor, G.P., Bickel, U., and Fehm, H.L. (2002) Sniffing neuropeptides: a transnasal approach to the human brain. Nature Neuroscience 5 514-516.

15. Bradbury, M.W.B., Cserr, H.F., and Westrop, R.J. (1981) Drainage of cer-ebral interstitialfluid into deep cervical lymph of the rabbit. American Jour-nal of Physiology 240 F329-F336.

16. Bradbury, M.W.B. and Westrop, R.J. (1983) Factors influencing exit of sub-stances from cerebrospinal fluid into deep cervical lymph of the rabbit. Journal of Physiology 339 519-534.

17. Brenneman, K.A., Wong, B.A., Buccellato, M.A., Costa, E.R., Gross, E.A., and Dorman, D.C. (2000) Direct olfactory transport of inhaled manga-nese ((MnCl2)-Mn-54) to the rat brain: Toxicokinetic investigations in a unilateral nasal occlusion model. Toxicology and Applied Pharmacology 169 238-248.

18. Broadwell, R.D. and Balin, B.J. (1985) Endocytic and exocytic pathways of the neuronal secretory process and trans-synaptic transfer of wheat germ agglutinin-horseradish peroxidase in vitro. The Journal of Comparative Neu-rology 242 632-650.

19. Burstein, A.H., Modica, R., Hatton, M., Forrest, A., and Gengo, F.M. (1997) Pharmacokinetics and pharmacodynamics of midazolam after intranasal administration. Journal of Clinical Pharmacology 37 711-718.

20. Calderon-Garciduenas, L., Azzarelli, B., Acuna, H., Garcia, R., Gambling, T.M., Osnaya, N., Monroy, S., Tizapantzi, M.D., Carson, J.L., Villarreal, C.A., and Rewcastle, B. (2002) Air pollution and brain damage. Toxicologic Pathology 30 373-389.

(27)

21. Capsoni, S., Giannotta, S., and Cattaneo, A. (2002) Nerve growth factor and galantamine ameliorate early signs of neurodegeneration in anti-nerve growth factor mice. Proceedings of the National Academy of Sciences of the United States of America 99 12432-12437.

22. Chen, X.Q., Fawcett, J.R., Rahman, Y.E., Ala, T.A., and Frey, W.H. (1998) Delivery of nerve growth factor to the brain via the olfactory pathway. Journal of Alzheimer’s Disease 1 35-44.

23. Chou, K.J. and Donovan, M.D. (1997) Distribution of antihistamines into the CSF following intranasal delivery. Biopharmaceutics and Drug Disposition 18 335-346.

24. Chou, K.J. and Donovan, M.D. (1998) Lidocaine distribution into the CNS following nasal and arterial delivery: a comparison of local sampling and microdialysis techniques. International Journal of Pharmaceutics 171 53-61. 25. Chou, K.J. and Donovan, M.D. (1998) The distribution of local anesthetics

into the CSF following intranasal administration. International Journal of Phar-maceutics 168 137-145.

26. Chou, R.C. and Levy, G. (1981) Effect of heparin or salicylate infusion on serum protein binding and on concentrations of phenytoin in serum, brain and cerebrospinal fluid of rats. Journal of Pharmacology and Experimental Thera-peutics 219 42-48.

27. Chow, H.H.S., Anavy, N., and Villalobos, A. (2001) Direct nose-brain trans-port of benzoylecgonine following intranasal administration in rats. Journal of Pharmaceutical Sciences 90 1729-1735.

28. Chow, H.H.S., Chen, Z., and Matsuura, G.T. (1999) Direct transport of cocaine from the nasal cavity to the brain following intranasal cocaine ad-ministration in rats. Journal of Pharmaceutical Sciences 88 754-758.

29. Chowdhury, S.I., Lee, B.J., Mosier, D., Sur, J.H., Osorio, F.A., Kennedy, G., and Weiss, M.L. (1997) Neuropathology of bovine herpesvirus type 5 (BHV-5) meningo-encephalitis in a rabbit seizure model. Journal of Comparative Pathology 117 295-310.

30. Cserr, H.F., Cooper, D.N., and Milhorar, T.H. (1977) Flow of cerebral in-terstitial fluid as indicated by the removal of extracellular markers from rat caudate nucleus. Experimental Eye Research Suppl 461-473.

(28)

32. Dahlin, M., Bergman, U., Jansson, B., Björk, E., and Brittebo, E. (2000) Transfer of dopamine in the olfactory pathway following nasal administra-tion in mice. Pharmaceutical Research 17 737-742.

33. Dahlin, M. and Björk, E. (2000) Nasal absorption of (S)-UH-301 and its transport into the cerebrospinal fluid of rats. International Journal of Pharma-ceutics 195 197-205.

34. Dahlin, M. and Björk, E. (2001) Nasal administration of a physostigmine analogue (NXX-066) for Alzheimer’s disease to rats. International Journal of Pharmaceutics 212 267-274.

35. Dahlin, M., Jansson, B., and Björk, E. (2001) Levels of dopamine in blood and brain following nasal administration to rats. European Journal of Phar-maceutical Sciences 14 75-80.

36. David, G.F.X., Puri, C.P., and Anand Kumar, T.C. (1981) Bioavailability of progesterone enhanced by intranasal spraying. Experientia 37 533-534. 37. Davson, H., Welch, K., and Segal, M.B. (1987) The secretion of the

cer-ebrospinal fluid. In: Physiology and Pathophysiology of the Cercer-ebrospinal Fluid, Churchill Livingstone, Edinburgh, 189-246.

38. De Souza Silva, M.A., Mattern, C., Hacker, R., Nogueira, P.J.C., Huston, J.P., and Schwarting, R.K.W. (1997) Intranasal administration of the dopaminergic agonists L-DOPA, amphetamine, and cocaine increases dopamine activity in the neostriatum: A microdialysis study in the rat. Jour-nal of Neurochemistry 68 233-239.

39. Denecke, H., Czehak, N., and Pietrowsky, R. (2002) Dose-response rela-tionships of intranasal cholecystokinin and the P300 event-related brain potential. Pharmacology Biochemistry and Behavior 73 593-600.

40. Derad, I., Willeke, K., Pietrowsky, R., Born, J., and Fehm, H.L. (1998) In-tranasal angiotensin II directly influences central nervous regulation of blood pressure. American Journal Of Hypertension 11 971-977.

41. DeSesso, J.M. (1993) The relevance to humans of animal models for inha-lation studies of cancer in the nose and upper airways. Quality Assurance 2 213-231.

42. Dluzen, D.E. and Kefalas, G. (1996) The effects of intranasal infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) upon catecholamine concentrations within olfactory bulbs and corpus striatum of male mice. Brain Research 741 215-219.

43. Dorman, D.C., Brenneman, K.A., McElveen, A.M., Lynch, S.E., Roberts,

(29)

K.C., and Wong, B.A. (2002) Olfactory transport: A direct route of deliv-ery of inhaled manganese phosphate to the rat brain. Journal of Toxicology and Environmental Health, Part A 65 1493-1511.

44. Dorman, D.C., Struve, M.F., James, R.A., Marshall, M.W., Parkinson, C.U., and Wong, B.A. (2001) Influence of particle solubility on the delivery of inhaled manganese to the rat brain: Manganese sulfate and manganese tetroxide pharmacokinetics following repeated (14-day) exposure. Toxicol-ogy and Applied PharmacolToxicol-ogy 170 79-87.

45. Draghia, R., Caillaud, C., Manicom, R., Parivani, A., Kahn, A., and Poenaru, L. (1995) Gene delivery into the central nervous system by nasal instilla-tion in rats. Gene Therapy 2 418-423.

46. Dufes, C., Olivier, J.C., Gaillard, F., Gaillard, A., Couet, W., and Muller, J.M. (2003) Brain delivery of vasoactive intestinal peptide (VIP) following nasal administration to rats. International Journal of Pharmaceutics 225 87-97. 47. Einer-Jensen, N., Larsen, L., Deprez, S., Starns, E., and Schwartz, S. (2001) Intranasal absorption of sumatriptan and naratriptan: no evidence of local transfer from the nasal cavities to the brain arterial blood in male rats. Biopharmaceutics and Drug Disposition 22 213-219.

48. Eriksson, C., Bergman, U., Franzén, A., Sjöblom, M., and Brittebo, E.B. (1999) Transfer of some carboxilic acids in the olfactory system following intranasal administration. Journal of Drug Targeting 7 131-142.

49. Erlich, S.S., McComb, J.G., Hyman, S., and Weiss, M.H. (1986) Ultrastruc-tural morphology of the olfactory pathway for cerebrospinal fluid drain-age in the rabbit. Journal of Neurosurgery 64 466-473.

50. Evans, J. and Hastings, L. (1992) Accumulation of Cd(II) in the CNS de-pending on the route of administration - intraperitoneal, intratracheal or intranasal. Fundamental and Applied Toxicology 19 275-278.

51. Faber, H.K. (1938) The early lesions of poliomyelitis after intranasal in-oculation. Journal of Pediatrics 13 10-37.

52. Faber, H.K. and Gebhardt, L.P. (1933) Localizations of the virus of polio-myelitis in the central nervous system during the preparalytic period, after intranasal instillation. Journal of Experimental Medicine 57 933-954.

53. Faber, W.M. (1937) The nasal mucosa and the subarachnoid space. Ameri-can Journal of Anatomy 62 121-148.

(30)

55. Fechter, L.D., Johnson, D.L., and Lynch, R.A. (2002) The relationship of particle size to olfactory nerve uptake of a non-soluble form of manga-nese into brain. Neurotoxicology 23 177-183.

56. Fehm, H.L., Perras, B., Smolnik, R., Kern, W., and Born, J. (2000) Manipu-lating neuropeptidergic pathways in humans: a novel appraoch to neurop-harmacology? European Journal of Pharmacology 405 43-54.

57. Fehm, H.L., Smolnik, R., Kern, W., McGregor, G.P., Bickel, U., and Born, J. (2001) The melanocortin melanocyte-stimulating hormone/adrenocor-ticotropin_4-10 decreases body fat in humans. The Journal of Clinical Endocrinology and Metabolism 86 1144-1148.

58. Frey, W.H., Liu, J., Chen, X., Thorne, R.G., Fawcett, J.R., Ala, T.A., and Rahman, Y. (1997) Delivery of 125-I-NGF to the brain via the olfactory route. Drug Delivery 4 87-92.

59. Frey, W.H., Liu, J., Thorne, R.G., and Rahman, Y.E. (1995) Intranasal deliv-ery of I-125-labeled nerve growth factor to the brain via the olfactory route. In: Research Advances in Alzheimer’s Disease and Related Disorders, Edi-tors: Iqbal, K., Mortimer, J.A., Winblad, B., and Wisniewski, H.M.; John Wiley & Son Ltd, Chichester, 329-335.

60. Gais, S., Sommer, M., Fischer, S., Perras, B., and Born, J. (2002) Post-trial administration of vasopressin in humans does not enhance memory for-mation (vasopressin and memory consolidation). Peptides 23 581-583. 61. Gianutsos, G., Morrow, G.R., and Morris, J.B. (1997) Accumulation of

manganese in rat brain following intranasal administration. Fundamental and Applied Toxicology 37 102-105.

62. Gizurarson, S., Thorvaldsson, T., Sigurdsson, P., and Gunnarsson, E. (1997) Selective delivery of insulin into the brain: intraolfactory absorption. Inter-national Journal of Pharmaceutics 146 135-141.

63. Gomes, N.G.L., Boni, M., and Primo, C.C. (1997) The invasive behaviour of cryptococcus neoformans: a possibility of direct acces to the central nervous system? Mycopathologia 140 1-11.

64. Gopinath, P.G., Gopinath, G., and Anand Kumar, T.C. (1978) Target site of intranasally sprayed substances and their transport across the nasal mucosa: A new insight into the intranasal route of drug delivery. Current Therapeutic Research 23 596-607.

65. Gozes, I., Bardea, A., Reshef, A., Zamostiano, R., Zhukovsky, S., Rubinraut, S., Fridkin, M., and Brenneman, D.E. (1996) Neuroprotective strategy for Alzheimer disease: Intranasal administration of a fatty neuropeptide.

(31)

ceedings of the National Academy of Sciences of the United States of America 93 427-432.

66. Gozes, I., Giladi, E., Pinhasov, A., Bardea, A., and Brenneman, D.E. (2000) Activity-dependent neurotrophic factor: Intranasal administration of femtomolar-acting peptides improve performance in a water maze. Journal of Pharmacology and Experimental Therapeutics 293 1091-1098.

67. Graff, C.L. and Pollack, G.M. (2003) P-glycoprotein attenuates brain up-take of substrates after nasal instillation. Pharmaceutical Research 20 1225-1230.

68. Harkema, J.R. (1991) Comparative aspects of nasal airway anatomy - Rel-evance to inhalation toxicology. Toxicologic Pathology 19 321-336.

69. Hastings, L. and Evans, J.E. (1991) Olfactory primary neurons as a route of entry for toxic agents into the CNS. Neurotoxicology 12 707-714. 70. Henriksson, J., Tallkvist, J., and Tjälve, H. (1997) Uptake of nickel into the

brain via the olfactory neurons in rats. Toxicology Letters 91 153-162. 71. Henriksson, J., Tallkvist, J., and Tjälve, H. (1999) Transport of manganese

via the olfactory pathway in rats: Dosage dependency of the uptake and subcellular distribution of the metal in the olfactory epithelium and the brain. Toxicology and Applied Pharmacology 156 119-128.

72. Henriksson, J. and Tjälve, H. (2000) Manganese taken up into the CNS via the olfactory pathway in rats affects astrocytes. Toxicological Sciences 55 392-398.

73. Henry, R.J., Ruano, N., Casto, D., and Wolf, R.H. (1998) A pharmacokinetic study of midazolam in dogs: nasal drops vs. atomizer administration. Pediatric Dentistry 20 321-326.

74. Hruz, P., Zechner, S., Heimberg, D., Hobi, V., Schonenberger, G.A., Scheffler, K., Muller, S.F., and Seifritz, E. (2001) Intranasal administration of delta sleep-inducing peptide increases P300. Journal of Clinical Psychop-harmacology 21 626-628.

75. Hunt, C.A., MacGregor, R.D., and Siegel, R.A. (1986) Engineering tar-geted in vivo drug delivery. I. The physiological and physiochemical prin-ciples governing opportunities and limitations. Pharmaceutical Research 3 333-344.

(32)

77. Illum, L. (1996) Nasal delivery. The use of animal models to predict per-formance in man. Journal of Drug Targeting 3 427-442.

78. Illum, L. (2000) Transport of drugs from the nasal cavity to the central nervous system. European Journal of Pharmaceutical Sciences 11 1-18.

79. Itaya, S.K. (1987) Anterograde transsynaptic transport of WGA-HRP in rat olfactory pathways. Brain Research 409 205-214.

80. Jackson, R.T., Tigges, J., and Arnold, W. (1979) Subarachnoid space of the CNS, nasal mucosa, and lymphatic system. Archives of Otolaryngology 105 180-184.

81. Jansson, B. and Björk, E. (2002) Visualization of in vivo olfactory uptake and transfer using fluorescein dextran. Journal of Drug Targeting 10 379-386. 82. Jarolim, K.L., McCosh, J.K., Howard, M.J., and John, D.T. (2000) A light microscopy study of the migration of naegleria fowleri from the nasal submucosa to the central nervous system during the early stage of primary amebic meningoencephalitis in mice. Journal of Parasitology 86 50-55. 83. Jin, K.L., Xie, L., Childs, J., Sun, Y.J., Mao, X.O., Logvinova, A., and

Greenberg, D.A. (2003) Cerebral neurogenesis is induced by intranasal ad-ministration of growth factors. Annals of Neurology 53 405-409.

84. Kao, H.D., Traboulsi, A., Itoh, S., Dittert, L., and Hussain, A. (2000) En-hancement of the systemic and CNS specific delivery of L-dopa by the nasal administration of its water soluble prodrugs. Pharmaceutical Research 17 978-984.

85. Kern, W., Born, J., Schreiber, H., and Fehm, H.L. (1999) Central nervous system effects of intranasally administered insulin during euglycemia in men. Diabetes 48 557-563.

86. Kristensson, K. and Olsson, Y. (1971) Retrograde axonal transport of pro-tein. Brain Research 29 363-365.

87. Kritas, S.K., Nauwynck, H.J., and Pensaert, M.B. (1995) Dissemination of wild-type and gC-, gE-and gI-deleted mutants of Aujeszky’s disease virus in the maxillary nerve and trigeminal ganglion of pigs after intranasal in-oculation. Journal of General Virology 76 2063-2066.

88. Krude, H., Biebermann, H., Schnabel, D., Tansek, M.Z., Theunissen, P., Mullis, P.E., and Gruters, A. (2003) Obesity due to proopiomelanocortin deficiency: Three new cases and treatment trials with thyroid hormone and ACTH4-10. Journal of Clinical Endocrinology and Metabolism 88 4633-4640.

(33)

89. Krzymowski, T., Grzegorzewski, W., Stefanczyk-Krzymowska S., Skipor, J., and Wasowska, B. (1999) Humoral pathway for transfer of the boar pheromone, androstenol, from the nasal mucosa to the brain and hypo-physis of gilts. Theriogenology 52 1225-1240.

90. Kumbale, R. (1999) GM1 delivery to the CSF via the olfactory pathway. Drug Delivery 6 23-30.

91. Larsson, P. and Tjälve, H. (2000) Intranasal instillation of aflatoxin B-1 in rats: Bioactivation in the nasal mucosa and neuronal transport to the olfac-tory bulb. Toxicological Sciences 55 383-391.

92. Lee, B.J., Weiss, M.L., Mosier, D., and Chowdhury, S.I. (1999) Spread of bovine herpesvirus type 5 (BHV-5) in the rabbit brain after intranasal in-oculation. Journal of Neurovirology 5 474-484.

93. Lindhardt, K., Gizurarson, S., Stefansson, S.B., Olafsson, D.R., and Bechgaard, E. (2001) Electroencephalographic effects and serum concen-trations after intranasal and intravenous administration of diazepam to healthy volunteers. British Journal of Clinical Pharmacology 52 521-527. 94. Lindquist, N.G., Lyden, A., Narfstrom, K., and Samaan, H. (1983)

Accu-mulation of taurine in the nasal mucosa and the olfactory bulb. Experientia 39 797-799.

95. Liu, X.F., Fawcett, J.R., Thorne, R.G., DeFor, T.A., and Frey, W.H. (2001) Intranasal administration of insulin-like growth factor-I bypasses the blood-brain barrier and protects against focal cerebral ischemic damage. Journal of the Neurological Sciences 187 91-97.

96. Madrid, M.Y. and Langer, R. (1991) Intranasal drug delivery to the central nervous system. Proceedings of the International Symposium of the Controlled Re-lease of Bioactive Materials 18 283-284.

97. Marra, A. and Brigham, D. (2001) Streptococcus pneumoniae causes ex-perimental meningitis following intranasal and otitis media infections via a nonhematogenous route. Infection and Immunity 69 7318-7325.

98. Martinez, A.J., Duma, R.J., Nelson, E.C., and Moretta, F.L. (1973) Experi-mental naegleria meningoencephalitis in mice. Penetration of the olfac-tory mucosal epithelium by naegleria and pathologic changes produced: a light and electron microscope study. Laboratory Investigation 29 121-133. 99. Mathison, S., Nagilla, R., and Kompella, U.B. (1998) Nasal route for direct

(34)

100. Meredith, M. and O’Connell, R.J. (1988) HRP uptake by olfactory and vomeronasal receptor neurons: use as an indicator of incomplete lesions and relevance for non-votile chemoreception. Chemical Senses 13 487-515. 101. Merkus, P., Guchelaar, H.J., Bosch, D.A., and Merkus, F.W.H.M. (2003)

Direct access of drugs to the human brain after intranasal drug adminis-tration? Neurology 60 1669-1671.

102. Mori, I., Goshima, F., Imai, Y., Kohsaka, S., Sugiyama, T., Yoshida, T., Yokochi, T., Nishiyama, Y., and Kimura, Y. (2002) Olfactory receptor neu-rons prevent dissemination of neurovirulent influenza A virus into the brain by undergoing virus-induced apoptosis. Journal of General Virology 83 2109-2116.

103. Morrison, E.E. and Costanzo, R.M. (1992) Morphology of olfactory epi-thelium in humans and other vertebrates. Microscopy Research and Techniques 23 49-61.

104. Murray, R.C. and Calof, A.L. (1999) Neuronal regeneration: Lessons from the olfactory system. Seminars in Cell and Developmental Biology 10 421-431. 105. Narita, M., Uchimura, A., Kawanabe, M., Fukushi, H., and Hirai, K. (2001)

Invasion and spread of equine herpesvirus 9 in the olfactory pathway of pigs after intranasal inoculation. Journal of Comparative Pathology 124 265-272.

106. Orosz, A., Földes, I., Kósa, Cs., and Tóth, G. (1957) Radioactive isotope studies of the connection between the lymph circulation of the nasal mu-cosa, the cranial cavity and cerebrospinal fluid. Acta Physiologica Academy of Science Hungary 2 75-81.

107. Öhman, L., Hahnenberger, R., and Johansson, E.D.B. (1980) 17b-Estradiol levels in blood and cerebrospinal fluid after ocular and nasal administra-tion in women and female rhesus monkeys (Macaca mulatta). Contracepadministra-tion 22 349-358.

108. Pardridge, W.M. (1996) Brain drug delivery and blood-brain barrier trans-port. Drug Delivery 3 99-115.

109. Park, C.H., Ishinaka, M., Takada, A., Kida, H., Kimura, T., Ochiai, K., and Umemura, T. (2002) The invasion routes of neurovirulent A Hong Kong 483/97 (H5N1) influenza virus into the central nervous system after respi-ratory infection in mice. Archives of Virology 147 1425-1436.

110. Pelidou, S.H., Zou, L.P., Deretzi, G., Nennesmo, I., Wei, L., Mix, E., van-der-Meide, P.H., and Zhu, J. (2000) Intranasal administration of recombinant

(35)

mouse interleukin-12 increases inflammation and demyelination in chronic experimental autoimmune neuritis in Lewis rats. Scandinavian Journal of Im-munology 51 29-35.

111. Perl, D.P. and Good, P.F. (1987) Uptake of aluminium into central nervous system along nasal-olfactory pathways. Lancet 1 1028.

112. Perlman, S., Evans, G., and Afifi, A. (1990) Effect of olfactory bulb abla-tion on spread of a neurotropic coronavirus into the mouse brain. Journal of Experimental Medicine 172 1127-1132.

113. Perras, B., Marshall, L., Köhler, G., Born, J., and Fehm, H.L. (1999) Sleep and endocrine changes after intranasal administration of growth hormone-releasing hormone in young and aged humans. Psychoneuroendocrinology 24 743-757.

114. Perras, B., Wagner, U., Born, J., and Fehm, H.L. (2003) Improvement of sleep and pituitary-adrenal inhibition after subchronic intranasal vasopressin treatment in elderly humans. Journal of Clinical Psychopharmacology 23 35-44. 115. Persson, E., Henriksson, J., Tallkvist, J., Rouleau, C., and Tjälve, H. (2003) Transport and subcellular distribution of intranasally administered zinc in the olfactory system of rats and pikes. Toxicology 191 97-108.

116. Persson, E., Henriksson, J., and Tjälve, H. (2003) Uptake of cobalt from the nasal mucosa into the brain via olfactory pathways in rats. Toxicology Letters 145 19-27.

117. Persson, E., Larsson, P., and Tjälve, H. (2002) Cellular activation and neu-ronal transport of intranasally instilled benzo(a)pyrene in the olfactory sys-tem of rats. Toxicology Letters 133 211-219.

118. Pietrowsky, R., Claassen, L., Frercks, H., Fehm, H.L., and Born, J. (2001) Time course of intranasally administered cholecystokinin-8 on central nerv-ous effects. Neuropsychobiology 43 254-259.

119. Pietrowsky, R., Struben, C., Molle, M., Fehm, H.L., and Born, J. (1996) Brain potential changes after intranasal vs intravenous administration of vasopressin: Evidence for a direct nose brain pathway for peptide effects in humans. Biological Psychiatry 39 332-340.

120. Pietrowsky, R., Thiemann, A., Kern, W., Fehm, H.L., and Born, J. (1996) A nose-brain pathway for psychotropic peptides: Evidence from a brain evoked potential study with cholecystokinin. Psychoneuroendocrinology 21 559-572.

(36)

122. Rao, V.M. and Stella, V.J. (2003) When can cyclodextrins be considered for solubilization purposes? Journal of Pharmaceutical Sciences 92 927-932. 123. Reiss, C.S., Plakhov, I.V., and Komatsu, T. (1998) Viral replication in

olfac-tory receptor neurons and entry into the olfacolfac-tory bulb and brain. Annual New York Academy of Sciences 30 751-761.

124. Sabin, A.B. and Olitsky, P.K. (1936) Influence of pathway of infection of pathology of olfactory bulbs in experimental poliomyelitis. Proceedings in Social Experimental Biology and Medicine 35 300-301.

125. Sakane, T., Akizuki, M., Taki, Y., Yamashita, S., Sezaki, H., and Nadai, T. (1995) Direct drug transport from the rat nasal cavity to the cerebrospinal fluid: The relation to the molecular weight of drugs. Journal of Pharmacy and Pharmacology 47 379-381.

126. Sakane, T., Akizuki, M., Yamashita, S., Nadai, T., Hashida, M., and Sezaki, H. (1991) The transport of a drug to the cerebrospinal fluid directly from the nasal cavity - The relation to the lipophilicity of the drug. Chemical and Pharmaceutical Bulletin 39 2456-2458.

127. Sakane, T., Akizuki, M., Yamashita, S., Sezaki, H., and Nadai, T. (1994) Direct drug transport from the rat nasal cavity to the cerebrospinal fluid: The relation to the dissociation of the drug. Journal of Pharmacy and Pharma-cology 46 378-379.

128. Sakane, T., Akizuki, M., Yoshida, M., Yamashita, S., Nadai, T., Hashida, M., and Sezaki, H. (1991) Transport of cephalexin to the cerebrospinal fluid directly from the nasal cavity. Journal of Pharmacy and Pharmacology 43 449-451.

129. Sakane, T., Yamashita, S., Yata, N., and Sezaki, H. (1999) Transnasal deliv-ery of 5-fluorouracil to the brain in the rat. Journal of Drug Targeting 7 233-240.

130. San Joaquin, V.H., Altshuler, G.P., Robinson, W., Marks, M.I., and Weber, A. (1983) Heamophilus influenzae bacteremia and meningitis in infant rabbit after intranasal inoculation. Journal of Laboratory and Clinical Medicine 102 939-946.

131. Sarkar, M.A. (1992) Drug metabolism in the nasal mucosa. Pharmaceutical Research 9 1-9.

132. Scherrmann, J.M. (2002) Drug delivery to brain via the blood-brain barrier. Vascular Pharmacology 38 349-354.

133. Sehgal, A. and David, G.F.X. (1980) Patterns of transfer of tritiated

(37)

gesterone into blood & cerebrospinal fluid of rhesus monkeys following diverse methods of administration. Indian Journal of Experimental Biology 18 707-708.

134. Seki, T., Sato, N., Hasegawa, T., Kawaguchi, T., and Juni, K. (1994) Nasal absorption of zidovudine and its transport to cerebrospinal fluid in rats. Biological and Pharmaceutical Bulletin 17 1135-1137.

135. Shi, J., Perry, G., Berridge, M.S., Aliev, G., Siedlak, S.L., Smith, M.A., LaManna, J.C., and Friedland, R.P. (2002) Labeling of cerebral amyloid beta deposits in vivo using intranasal basic fibroblast growth factor and serum amyloid P component in mice. Journal of Nuclear Medicine 43 1044-1051.

136. Shinya, K., Shimada, A., Ito, T., Otsuki, K., Morita, T., Tanaka, H., Takada, A., Kida, H., and Umemura, T. (2000) Avian influenza virus intranasally inoculated infects the central nervous system of mice through the general visceral afferent nerve. Archives of Virology 145 187-195.

137. Shinya, K., Silvano, F.D., Morita, T., Shimada, A., Nakajima, M., Ito, T., Otsuki, K., and Umemura, T. (1998) Encephalitis in mice inoculated intranasally with an influenza virus strain originated from a water bird. Journal of Veterinay Medicinal Sciences 60 627-629.

138. Shipley, M.T. (1985) Transport of molecules from nose to brain: transneuronal anterograde and retrograde labeling in the rat olfactory sys-tem by wheat germ agglutinin-horseradish peroxidase applied to the nasal epithelium. Brain Research Bulletin 15 129-142.

139. Shipley, M.T. and Ennis, M. (1996) Functional organization of olfactory system. Journal of Neurobiology 30 123-176.

140. Sigurdsson, P., Thorvaldsson, T., and Gizurarson, S. (1997) Olfactory ab-sorption of insulin to the brain. Drug Delivery 4 195-200.

141. Smolnik, R., Molle, M., Fehm, H.L., and Born, J. (1999) Brain potentials and attention after acute and subchronic intranasal administration of ACTH 4-10 and desacetyl-alpha-MSH in humans. Neuroendocrinology 70 63-72. 142. Snyder, W.S., Cook, M.J., Nasset, E.S., Karhausen, L.R., Parry Howells, G.,

and Tipton, I.H. (1975) Report of the task group on reference man, edition 1, Pergamon press, Exeter.

(38)

144. Stewart, W.B. (1985) Labelling of olfactory bulb glomeruli following horse-radish peroxidase lavage of the nasal cavity. Brain Research 347 200-203. 145. Subramaniam, R.P., Richardson, R.B., Morgan, K.T., and Kimbell, J.S. (1998)

Computational fluid dynamics simulations of inspiratory airflow in the human nose and nasopharynx. Inhalation Toxicology 10 91-120.

146. Sunderman, F.W. (2001) Review: Nasal toxicity, carcinogenicity, and olfac-tory uptake of metals. Annals of Clinical and Laboraolfac-tory Science 31 3-24. 147. Tallkvist, J., Persson, E., Henriksson, J., and Tjälve, H. (2002)

Cadmium-metallothionein interactions in the olfactory pathways of rats and pikes. Toxicological Sciences 67 108-113.

148. Thorne, R.G., Emory, C.R., Ala, T.A., and Frey, W.H. (1995) Quantitative analysis of the olfactory pathway for drug delivery to the brain. Brain Re-search 692 278-282.

149. Tjälve, H. and Henriksson, I. (1999) Uptake of metals in the brain via olfactory pathways. Neurotoxicology 20 181-195.

150. Tjälve, H., Henriksson, J., Tallkvist, J., Larsson, B.S., and Lindquist, N.G. (1996) Uptake of manganese and cadmium from the nasal mucosa into the central nervous system via olfactory pathways in rats. Pharmacology and Toxi-cology 79 347-356.

151. Tomlinson, A.H. and Esiri, M.M. (1983) Herpes simplex encephalitis. Im-munohistological demonstration of spread of virus via olfactory pathways in mice. Journal of the Neurological Sciences 60 473-484.

152. Volkow, N.D., Wang, G.J., Fischman, M.W., Foltin, R., Fowler, J.S., Franceschi, D., Franceschi, M., Logan, J., Gatley, S.J., Wong, C., Ding, Y.S., Hitzemann, R., and Pappas, N. (2000) Effects of route of administration on cocaine induced dopamine transporter blockade in the human brain. Life Sciences 67 L1507-L1515.

153. Wang, F., Jiang, X., and Lu, W. (2003) Profiles of methotrexate in blood and CSF following intranasal and intravenous administration to rats. Inter-national Journal of Pharmaceutics 263 1-7.

154. Wang, H.W. and Su, W.Y. (1987) Retrograde axonal transport of true blue dye by the peripheral autonomic nerves in canine nasal mucosa. Archives of Otolaryngology 244 295-299.

155. Wang, Y., Aun, R., and Tse, F.L.S. (1998) Brain uptake of dihydroergotamine after intravenous and nasal administration in the rat. Biopharmaceutics and Drug Disposition 19 571-575.

(39)

156. Waynforth, H.B. and Flecknell, P.A. (1980) Cisternal puncture (and intrac-isternal injection). In: Experimental Techniques in the Rat, Academic Press, London, 59-61.

157. Weiner, H.L., Lemere, C.A., Maron, R., Spooner, E.T., Grenfell, T.J., Mori, C., Issazdeh, S., Hancock, W.W., and Selkoe, D.J. (2000) Nasal administra-tion of amyloid-beta peptide decreases cerebral amyloid burden in a mouse model of Alzheimer’s disease. Annals Of Neurology 48 567-579.

158. Yajima, T., Juni, K., Saneyoshi, M., Hasegawa, T., and Kawaguchi, T. (1998) Direct transport of 2',3'-didehydro-3'-deoxythymidine (D4T) and its ester derivatives to the cerebrospinal fluid via the nasal mucous membrane in rats. Biological and Pharmaceutical Bulletin 21 272-277.

159. Yoffey, J.M. (1958) Passage of fluid and other substances through the na-sal mucosa. Journal of Laryngology and Otology 72 377-383.

160. Youngentob, S.L., Schwob, J.E., Saha, S., Manglapus, G., and Jubelt, B. (2001) Functional consequences following infection of the olfactory system by intranasal infusion of the olfactory bulb line variant (OBLV) of mouse hepatitis strain JHM. Chemical Senses 26 953-963.

161. Yu, I.J., Park, J.D., Park, E.S., Song, K.S., Han, K.T., Han, J.H., Chung, Y.H., Choi, B.S., Chung, K.H., and Cho, M.H. (2003) Manganese distribu-tion in brains of Sprague-Dawley rats after 60 days of stainless steel weld-ing-fume exposure. Neurotoxicology 24 777-785.

(40)

(41)

(42)

2 Serial cerebrospinal fluid sampling in a rat

model to study drug uptake from the nasal

cavity

(43)

Abstract

Drug transport from the nasal cavity to the brain has gained high interest in the last decade. In the present study a model was developed to deter-mine the uptake of drugs into the cerebrospinal fluid (CSF) after nasal delivery in rats. CSF samples were taken using a cisternal puncture method. In this method a needle is advanced through the skin and muscles over-lying the atlanto-occipital membrane into the cisterna magna, while the rat is fixed in a stereotaxic frame. This method appeared to be superior over cannulation of the atlanto-occipital membrane for CSF sampling. The major advantages of the puncture method are the ability of serial and simultaneous CSF and blood sampling for over two hours in the same rat.

To obtain maximal drug absorption from the nasal cavity and uptake into CSF, different positions of the rat’s head (upright-90° angle, supine-90° angle, supine-45° angle and supine-70° angle) were tested in nasal deliv-ery studies using hydrocortisone (HC) as model drug. Putting the rat in the supine-90° angle position increased the absorption of HC into plasma and CSF two-fold compared to the upright-90° angle position. The su-pine-70° angle position did not change the HC plasma and CSF levels compared to the supine-90° angle position. However, the supine-70° angle position showed the fastest CSF sampling rate, enabling more accurate CSF sampling and therefore preferred for further studies.

In conclusion, the cisternal puncture method using the supine-70° and 90° angle position is a suitable method to study drug transport from the nasal cavity into the CSF, with the ability of multiple CSF sampling.

Keywords

(44)

nose-1. Introduction

Drug transport from the nasal cavity to the brain has been extensively investigated in the last decade (15, 19). This route gained much interest because it has major advantages over conventionally used delivery routes as the oral and intravenous ones, especially with regard to brain targeting of drugs. Firstly, the nose-brain route is not obstructed by the blood-brain barrier, which is one of the major problems in delivery of drugs to the central nervous system. A direct route from the nasal cavity to the brain also circumvents the first-pass elimination by the liver and gastrointestinal tract. For drugs that are currently administered by parenteral injections, the nose-brain route will enhance the patient com-pliance.

To study nose-to-brain transport of drugs properly it is important to choose a good model. To investigate whether a drug is delivered to the brain and/or cerebrospinal fluid (CSF) rather than to the systemic circu-lation, the drug uptake in brain tissue and/or CSF and blood needs to be monitored. For studying drug uptake into biological tissues and fluids requires an animal model, since the use of cell cultures represents only one body compartment at the time and can not simulate absorption and elimination of a compound. The choice of the species to be used should be made in such a way, that it could be related to the human situation. On guidance of these considerations an animal model to study drug up-take from the nasal cavity into the CSF should ideally meet the following conditions: 1) serial blood sampling over a certain period of time, 2) serial CSF sampling over that same period, 3) use of a species that has a close analogy to the human nose-brain transport route, and because of working with laboratory animals: 4) act according to the three R’s of Russell and Burch: Replacement, Reduction and Refinement (23). With respect to the third condition, a few studies concerning drug up-take into the brain after nasal delivery were performed in monkeys (1, 11) which have a nasal cavity showing close homology to humans (10). How-ever, most studies investigating nose-to-CSF drug delivery were done in rats (4, 6, 24, 26). Therefore, in the present studies the rat was chosen to

(45)

set-up as an experimental animal model. This rat model can be used to screen for potential nose-to-CSF transport of drugs. Subsequently, these drugs should be tested in species with closer analogy to humans, like rabbits or monkeys (10). The last condition (i.e. the three R’s) requires that the experimental set-up should have the least possible degree of discomfort for the animals. This has important implications for choos-ing the samplchoos-ing methods needed for the first two conditions (i.e. serial blood and CSF sampling).

(46)

for at least one week. Another CSF sampling method that meets the above mentioned criteria is the cisternal puncture method using a stereotaxic frame as described by Frankmann (9). In this method CSF samples are taken from the cisterna magna by putting a needle through the muscle layer overlying the a-o membrane and finally into the cisternal compart-ment while the anaesthetised rat is fixed in a stereotaxic frame, which is not invasive like the first mentioned cisternal puncture procedure. These two CSF sampling methods are already a refinement of the earlier men-tioned cisternal cannulation (2, 16, 17, 25, 27, 30) and puncture (4, 5, 29) methods.

The aim of the present study was to develop a suitable rat model for investigating drug transport from the nasal cavity to the CSF, which ful-fils all four above-mentioned criteria. To that purpose, the cannulation of the a-o membrane and the cisternal puncture using a stereotaxic frame were compared with each other, using the steroid hormone hydrocorti-sone (HC) as a model drug. Corticosteroids are involved in brain proc-esses like learning and memory, and may also be useful in the treatment of neurodegenerative diseases (18), making these compounds very inter-esting for studying drug targeting from the nasal cavity to the CSF.

2. Materials and methods

2.1 Materials

Hydrocortisone (HC) (at least 98 % purity) was from Sigma Chemical (St. Louis, MO, USA) and randomly methylated beta-cyclodextrin (RAMEB; degree of substitution 1.8) was from Wacker-Chemie (Krommenie, The Netherlands). Ethanol (96 %) of analytical grade was from Merck (Darmstadt, Germany) and sterile saline (0.9 % NaCl) was from the Hos-pital Pharmacy of Leiden University Medical Centre (Leiden, The Neth-erlands). Hypnorm®_{(fentanyl citrate 0.315 mg/ml, fluanisone 10 mg/} ml) was supplied by Janssen Pharmaceutica (Beerse, Belgium). Midazolam (5 mg/ml) was from Genthon B.V. (Nijmegen, the Netherlands). Nemb-utal®_{(pentobarbital sodium, 60 mg/ml) was purchased from Sanofi Sante} Nutrition Animale (Libourne, France) and Temgesic® _{(buprenorfine, 0.3} mg/ml) from Schering-Plough (Maarssen, The Netherlands). Collagenase

(47)

(455 U/mg digestion activity) was obtained from Sigma (St. Louis, MO, USA) and the tissue glue Histoacryl blue®_{was from B Braun (Melsungen,} Germany). All other reagents were of analytical grade.

2.2 Hydrocortisone formulations

HC and RAMEB were dissolved in ethanol in a molar ratio of 1:1.2 to generate inclusion complexes. RAMEB was used as solubiliser to en-hance the solubility of HC by inclusion complex formation. After evapo-ration of ethanol at 35°C under a mild nitrogen stream, the residues were dissolved in sterile saline to obtain two formulations with the following concentrations HC (mg/ml) and RAMEB (%, w/v), respectively: 7.0 mg/ ml and 3.0 %; 14.0 mg/ml and 6.0 %.

2.3 Animals

Male Wistar rats (Charles River, Someren, The Netherlands) were used, weighing 250 – 400 g at the start of the experiments. The animals were housed separately with free access to food and water with a 12-h light/ dark cycle. At the end of the experiments the animals were euthanised with an overdose of Nembutal®_{(1 – 2 ml, intraperitoneally).}

2.4 Cannulation of the atlanto-occipital membrane for CSF sampling