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NEUROPHARMACOLOGY
Oral Anatomy and Developmental Biology, School of Dentistry, Showa University, Tokyo, Japan (N.N.); First Anatomy, School of Medicine, Showa University, Tokyo, Japan (N.N., H.K., S.S.); and Geriatric Research, Education, and Clinical Center, Veterans Affairs Medical Center-St. Louis and Division of Geriatrics, Department of Internal Medicine, Saint Louis University School of Medicine, St. Louis, Missouri (N.N., S.A.F., W.A.B.)
Received September 30, 2007; accepted February 11, 2008.
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Abstract |
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-cyclodextrin, and the OB after i.n. administration with dimethyl-β cyclodextrin. These studies show that intranasal administration is an effective route of administration for the delivery of GALP to the brain and that targeting among brain regions may be possible with the use of various cyclodextrins.
). It acts at the three galanin receptors (GALR1, GALR2, and GALR3), with actions primarily mediated through GALR2 and probably another unidentified receptor (Branchek et al., 2000). GALP was originally cloned from pig hypothalamus (Ohtaki et al., 1999), and its expression is to some extent regulated by leptin (Gundlach, 2002). When administered by i.c.v. injection, GALP reduces food intake and body weight of the mouse (Krasnow et al., 2003).
There is interest in developing GALP as a therapeutic for the treatment of obesity (Gundlach, 2002). Such a use of GALP would require its peripheral administration and negotiation of the blood-brain barrier (BBB). GALP crosses the BBB at a modest rate through a saturable mechanism (Kastin et al., 2001). The transport rate of GALP is modulated by fasting and glucose administration, but it is unaffected by leptin. However, GALP has a short half-life in blood after i.v. administration making it difficult to deliver therapeutic doses of GALP by i.v. administration. Thus, development of more stable analogs, use of a delivery system that improves the pharmacokinetics of native GALP, or an alternate route of administration that bypasses blood enzymes is needed.
One alternate pathway for delivery of drugs to the brain is by i.n. administration (Frey, 2002). Some substances delivered to the cribriform plate are able to enter the brain by way of the olfactory nerves, which are direct extensions of the central nervous system. From here, they most generally are taken up by the OB (Baker and Spencer, 1986), but they can also enter into other areas of the brain by mechanisms that have been partially elucidated (Balin et al., 1986). For example, horseradish peroxidase leaks between the intercellular spaces of the nasal epithelium, and it uses extraneuronal pathways to reach deep areas of the brain in mouse (Balin et al., 1986), whereas uptake of an exendin analog is dependent on an energy-requiring process (Banks et al., 2004).
Intranasal administration holds some advantages for delivery of peptides, especially peptides with unfavorable pharmacokinetics after i.v. administration (Born et al., 2002; Frey, 2002). For example, insulin delivered to the brain by i.n. administration can avoid the hypoglycemia of i.v. administration (Kern et al., 1999) in amounts that promote learning in humans (Benedict et al., 2004). Glucagon-like peptide-1 is rapidly degraded in blood, but it or its antagonist can be given by i.n. administration in amounts that exert cognitive and neuroprotective effects (During et al., 2003; Banks et al., 2004). Other peptides shown to be taken up in significant amounts include leptin (Kastin and Pan, 2006) and vasoactive intestinal peptide (Dufes et al., 2003).
Although i.n. delivery shows great promise, little work has yet been done that compares the relative uptake or brain distribution after i.v., i.n., or i.c.v. administration. In addition, compounds are often given with cyclodextrins to enhance uptake. Cyclodextrins are cyclic glucans that can form inclusion complexes with many substances, thus increasing solubility and absorption. Three general categories are -, β-, and 
-cyclodextrins that consist of six, seven, or eight glucopyranose units, respectively. In vitro models of the BBB suggest that there are no differences in permeability among these three classes of cyclodextrins (Monnaert et al., 2004). However, no work has yet compared the relative effects of these various cyclodextrins on uptake of peptides administered by the i.n. route.
Here, we investigated the distribution of I-GALP into whole brain, olfactory bulb, and other selected brain regions, blood, cervical lymph nodes, and spleen after i.n., i.v., and i.c.v. administration. We also compared the effects of selected cyclodextrins on the uptake and distribution within brain of GALP administered by the i.n. route.
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Materials and Methods |
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Measurement of BBB Permeability to i.v. I-GALP. Male ICR mice (2 months old; 35
45 g) from our in-house colony were anesthetized with an i.p. injection of 0.2 ml of urethane (40% solution), and the right carotid artery and left jugular vein were exposed. Mice were given 200 µl of lactated Ringer's solution (LR) containing 1% bovine serum albumin (BSA) and 300,000 cpm of I-GALP by injection into the jugular vein. Blood was collected from the right carotid artery, and the whole brain (WBr) was removed at 1, 2, 3, 4, 5, 7.5, or 10 min after the i.v. injection of I-GALP. The WBr was weighed. The whole blood was centrifuged at 5400g for 15 min at 4°C, and the level of radioactivity was measured in 50 µl of the resulting serum. The level of radioactivity for WBr was also counted in a gamma counter for 3 min. The brain/serum ratios for WBr were calculated, and the unidirectional influx rate (Ki, in units of microliters per gram-minute), and initial volume of distribution in brain at time 0 (Vi, in units of microliters per gram) was determined by multipletime regression analysis with the following formula:
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In other mice, the cervical lymph nodes and spleen were identified, removed, and weighed. The brain was dissected on ice into the OB, anterior one third of the brain (ant brain), hippocampus, hypothalamus, cerebellum, and brain stem, and each region was weighed. The whole blood was centrifuged at 5400g for 15 min at 4°C, and the level of radioactivity was measured in 50 µl of the resulting serum. The levels of radioactive iodine in the serum and tissue samples were determined by counting in a gamma counter for 30 min. The percentage of the injected dose taken up per gram of brain tissue (%Inj/g) was calculated at each time with the following formula:
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Intranasal Administration. Male ICR mice (2 months old; 3545 g) from our in-house colony were anesthetized with an i.p. injection of 0.2 ml of urethane (40% solution), and the right carotid artery was exposed. Mice were given an i.n. administration of 2 µlof 250,000 cpm/µl I-GALP alone, I-GALP mixed with dimethyl-β-cyclodextrin (I-GALP + B-CD) (Sigma-Aldrich, St. Louis, MO), I-GALP mixed with -cyclodextrin (I-GALP + A-CD) (Sigma-Aldrich), or I-GALP with 1 µg/mouse of unlabeled GALP. A total of 2 µl was delivered to the cribriform plate by pushing a small cannula attached to a 10-µl syringe through the right nares to the depth of the cribriform plate. The I-GALP + cyclodextrin (CD) solutions were prepared by mixing equal volumes of I-GALP with a 20% CD solution. Blood was collected from the right carotid artery, and the whole brain was removed at 1, 2, 3, 4, 5, 7.5, or 10 min after i.n. administration. The mouse was decapitated, and the cervical lymph nodes were identified, removed, and weighed. The brain was dissected on ice into OB, ant brain, hippocampus, hypothalamus, cerebellum, and brain stem, and each region was weighed. The whole blood was centrifuged at 5400g for 15 min at 4°C, and the level of radioactivity measured in 50 µl of resulting serum. The level of radioactive iodine in the serum and tissue samples was determined by counting in a gamma counter for 30 min. Results are expressed as %Inj/g.
Intracerebroventricular Injection. Male ICR mice (2 months old; 3545 g) from our in-house colony were anesthetized with an i.p. injection of 0.2 ml of urethane (40% solution). For each mouse, the skin was removed from over the skull, and a hole made into the lateral ventricle of the brain (1.0 mm lateral and 0.5 mm posterior to the bregma) with a 26-gauge needle. Tubing covered all but the terminal 3.0 to 3.5 mm of the needle so as to make a track through the brain and into the lateral ventricle but not through the floor of the lateral ventricle. The mice were given an injection of 1.0 µl of LR containing 20,000 cpm of I-GALP into the lateral ventricle of the brain. Blood was collected from the right carotid artery, and the WBr was removed at 10 min after an i.c.v. injection. The mouse was decapitated, and the cervical lymph nodes and spleen were identified, removed, and weighed. The brain was dissected on ice in to OB, ant brain, hippocampus, hypothalamus, cerebellum, and brain stem, and the regions were weighed. The whole blood was centrifuged at 5400g for 15 min at 4°C, and the level of radioactivity measured in 50 µl of resulting serum. The level of radioactive iodine in the serum and tissue samples was determined by counting in a gamma counter for 30 min. The results are expressed as %Inj/g.
Cerebrospinal Fluid Collection. Male ICR mice (2 months old; 3545 g) from our in-house colony were anesthetized with an i.p. injection of 0.2 ml of urethane (40% solution). The left jugular vein and right carotid artery were exposed. The mice were given an i.v. injection into the left jugular vein of 0.2 ml of 1% BSA in LR containing 300,000 cpm of I-GALP. Other mice were given an i.n. administration of 2 µl of 250,000 cpm/µl of I-GALP + B-CD. A total of 2 µl was delivered to the cribriform plate by pushing a small cannula attached to a 10-µl syringe through the right nares to the depth of the cribriform plate. Five minutes later, the skin overlying the posterior fossa was removed, and a 30-gauge needle connected to polyethylene-20 tubing was pushed into the posterior fossa. CSF was allowed to passively fill the tubing, and the amount of CSF collected was determined by measuring the length in centimeters of the polyethylene-20 tubing filled with CSF and multiplying by 1.134 (the volume of the tubing being 1.134 µl/cm). Only CSF that was absolutely clear was analyzed. Blood was collected from the right carotid artery, and the whole brain was removed and weighed after i.n. or i.v. administration. The mouse was decapitated, and then the OB and other brain regions were dissected on ice from the whole brain, and each region was weighed. The whole blood was centrifuged at 5400g for 15 min at 4°C, and the level of radioactivity was measured in 50 µl of resulting serum. The level of radioactive iodine in the serum and tissue samples were determined by counting in a gamma counter for 30 min. Values for whole brain were calculated by adding the regional weights and regional levels of radioactivity. The %Inj/ml was calculated with the following formula:
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Acid Precipitation. Male ICR mice (2 months old; 3545 g) were anesthetized, prepared, and administered I-GALP by the i.n., i.c.v., or i.v. route. For i.n. administration, I-GALP was given with B-CD. Blood was collected from the right carotid artery, and the OB and WBr were removed at 5 and 10 min (n = 36 mice/time) after i.n., i.c.v., or i.v. administration of I-GALP. The OB and WBr were dissected from brain on ice, and 150 µl of 0.25 M phosphate-buffered solution and 150 µl of 10 mM enzymatic cocktail (19.82 mg/ml 100 mM 1,10-phenanthroline, 12.51 mg/ml 100 mM N-ethylmaleimide, 37.22 mg/ml 100 mM EDTA, and 88.89 mg/ml 100 mM L-thyroxine in distilled water; Sigma-Aldrich) were added to the OB or WBr. The samples were homogenized on ice with a Tissue Tearor (Biospec Products, Inc., Racine, WI) for 20 s at a setting of 20. The homogenate was centrifuged at 5400g for 15 min. A portion of the supernatant (150 µl) was transferred to a second tube, and 150 µl of 30% trichloroacetic acid (TCA) (Sigma-Aldrich) was added to precipitate the I-GALP. Whole blood was centrifuged at 5400g for 15 min at 4°C, and the level of radioactivity was measured in 50 µl of the resulting serum. Ten microliters of serum was added to a tube containing 500 µl of 1% BSA in LR, the contents were mixed, and 500 µl of 30% TCA was added. This combination was then mixed and centrifuged. To determine the amount of degradation that occurred during this processing, 500,000 cpm of I-GALP was added in vitro to serum, OB, or WBr obtained from a mouse that had not received an i.n., i.c.v., or i.v. administration (n = 2 mice). Samples were then processed, homogenized, and centrifuged as described above. After addition of TCA, the serum, brain, and processing controls were centrifuged, and the supernatant was separated from the pellet. The percentage of radioactivity precipitated was calculated by the following formula:
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Statistical Analysis. The Prism 4.0 program (GraphPad Software Inc., San Diego, CA) was used in statistical analysis. Regression lines were computed by the least-squares method in Prism. Means are reported with their n and S.E., and they were compared by analysis of variance (ANOVA) followed by Newman-Keuls range test.
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Results |
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6 mice/region). The amount of radioactivity that precipitated with acid varied little between the 5- and 10-min values, but a two-way ANOVA did show variations among administration routes [F(5,76) = 5.19; p < 0.001], regions [F(2,76) = 14.7; p < 0.001], and interactions between routes and regions [F(10,76) = 5.83; p < 0.001]. Newman-Keuls tests showed the following differences at p < 0.05: WBr INA at 5 min was less than i.c.v. or i.v. WBr at 5 min and i.v. WBr at 10 min was higher than i.c.v. or i.n. WBr at 10 min.
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We measured the rate of unidirectional influx (Ki) from blood-to-brain for I-GALP administered by i.v. injection. Figure 1 shows the relation between whole brain/serum ratio (microliters per gram) and exposure time (minutes). Ki for I-GALP into brain was 0.90 ± 0.35 µl/g-min (r = 0.58, p < 0.05, n = 15 mice). Equilibrium between brain and blood was reached approximately 5 min after injection. The Vi was 11.0 ± 1.4 µl/g, a space about equal to the vascular volume of the brain.
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Uptake into cerebrospinal fluid was measured after i.n. administration of I-GALP + B-CD and after i.v. injection of I-GALP into each of three mice (Fig. 2). WBr, OB, and CSF were collected 5 min later. CSF had the highest level of I-GALP of any region after i.v. administration, but olfactory bulb had the highest level after i.n. administration. A two-way ANOVA was significant for route [F(1,12) = 15.0; p < 0.005] and interaction [F(2,12) = 4.29; p < 0.05], but not for region. Newman-Keuls analysis showed that I-GALP was higher in the OB after i.n. administration than in the OB or WBr after i.v. administration. CSF values did not differ between the i.n. and the i.v. routes.
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The time-dependent uptake of I-GALP + B-CD by whole brain, olfactory bulb, cervical lymph nodes, and spleen was further explored (Fig. 4). Entry of I-GALP + B-CD into the olfactory bulb after i.n. administration (n = 3 mice/time point) was higher than into any of the other tissues (Fig. 4A). After i.v. injection, I-GALP uptake (n = 3 mice/time point) by spleen was higher than by any another tissue, and WBr had the lowest uptake (Fig. 4B). Figure 4C compares these results for the brain regions of WBr and OB after i.n. or i.v. administration. Entry into OB was higher than uptake by WBr after i.n. or i.v. injection, and i.n. injection produced higher levels than did i.v. for either OB or WBr uptake. Figure 4D compares these results for the cervical lymphatic nodes and the spleen after i.n. or i.v. administration. Entry of I-GALP into the spleen after i.v. administration was higher than into the cervical lymph nodes or spleen after i.n. administrations or into the cervical lymph nodes after i.v. administration. The i.v. injection produced higher levels in spleen than did the i.n. injection.
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15 mice) (Fig. 5). Hypothalamus was again higher than any other tissue after i.n. administration of I-GALP (Fig. 5A). OB and hypothalamus were much higher than any other brain regions after i.n. administration of I-GALP + CD (Fig. 5B). Thus, A-CD results were distinct from those with either I-GALP alone or with B-CD. Every brain region after i.n. administration of I-GALP + A-CD was higher than after i.n. administration of I-GALP without CD.
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Discussion |
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). Equilibrium between WBr and blood was reached approximately 5 min after i.v. injection. When expressed as %Inj/g, steady state was reached in WBr, OB, spleen, and cervical lymphatics within approximately 1 min of i.v. injection and within 1 to 5 min after i.n. administration. I-GALP was found by acid precipitation to be stable in blood, OB, and brain after i.v., i.n., or i.c.v. administration. I-GALP may have been somewhat less stable in WBr after i.n. administration compared with the other compartments and routes of administration.
In contrast to the similarity between i.n. and i.v. routes for time to equilibrium, stability in tissues, and also CSF uptake, the distributions in CNS and peripheral tissues differed markedly among the three routes of administration. In general, brain uptake was 5 to 10 times higher after i.n. than after i.v. administration of I-GALP. OB uptake was particularly high after i.n. administration, consistent with its entry into the CNS at the cribriform plate. High OB uptake is typical with i.n. administration, especially in rodents, which have very large OB. Otherwise, distribution throughout the rest of the brain was rather uniform after i.n. administration. In contrast, the cerebellum had the highest uptake after i.v. administration, with olfactory bulb having the second highest uptake. It is possible that the saturable transporter located at the BBB is most active in these brain regions. Hippocampus, an area of the brain with receptors for GALP was an area of intermediate uptake after i.v. injection, but the area with the second highest uptake after i.c.v. administration. The high uptake for hippocampus and hypothalamus after i.c.v. injection may reflect the proximity these tissues have to the lateral ventricle, whereas after i.v. injection, it may be that receptor density is the main determinant.
The relative uptakes by the peripheral tissues of the cervical lymph nodes and the spleen are consistent with the access of these tissues according to the route of administration. After i.v. administration, I-GALP has equal access to the cervical nodes and spleen by way of the systemic circulation. Thus, splenic and cervical lymph node levels of I-GALP approximate each other. After i.c.v. administration, I-GALP would enter the circulation primarily with the reabsorption of cerebrospinal fluid into the cervical lymphatics (Widner et al., 1987; Boulton et al., 1999). This would give I-GALP immediate access to the (Yamada et al., 1991; Knopf et al., 1995) cervical lymph nodes, whereas access to the spleen would be dependent on lymphatic drainage into the circulation. Thus, uptake of I-GALP by the cervical lymph nodes is much higher than uptake by the spleen after i.c.v. administration. The distribution pattern between cervical lymph nodes and spleen is similar for i.n. and i.c.v. administered I-GALP, consistent with i.n.-administered I-GALP using the same pathways as i.c.v. administered I-GALP to enter the circulation. However, levels of I-GALP in spleen and cervical lymph nodes were much lower after i.n. than after i.c.v. administration, indicating that much less I-GALP effluxed from brain to blood after i.n. than after i.c.v. administration. These findings have two relevant considerations. First, drainage from the CNS to the cervical lymph nodes has been associated with modulation of neuroimmune events (Cserr and Knopf, 1992; Knopf et al., 1995) and uptake from brain to blood of peptides can modulate splenic function (Martins et al., 1997). Our findings show that each of the three routes would produce a very different profile of neuroimmune versus peripheral immune activation. Second, the spleen serves as a proxy or indicator of the extent to which peripheral tissues are exposed to substances delivered i.v. versus i.n. Thus, after i.v. injection, the %Inj/g for whole brain is less than 1/10 of that for spleen, but after i.n. injection, whole brain is approximately twice that for spleen. This shows that the delivery of I-GALP to the CNS relative to its peripheral delivery is approximately 20 times more after i.n. than after i.v. injection. This compares with an approximately 7-fold favoring of the CNS versus periphery for an exendin antagonist (Banks et al., 2004). Such directionality shows that i.n. delivery of peptides could produce more CNS effects while reducing unwanted peripheral effects.
We compared the influence of cyclodextrins on nasal absorption of I-GALP. Most of these studies (e.g., Fig. 3) were performed with dimethyl-β cyclodextrin, but in Fig. 5, we compare I-GALP administered i.n. without cyclodextrin or with -cyclodextrin. Both cyclodextrins tended to increase uptake of I-GALP to all regions of the brain. It is interesting to note that I-GALP administered without cyclodextrin showed the highest uptake by the hypothalamus, not the OB. This could indicate that the presence of receptors for GALP rather than anatomical proximity to the region of administration was the most influential factor in the absence of cyclodextrin. The addition of -cyclodextrin increased uptake by all regions of the brain by approximately 2-fold, with the exception for the olfactory bulb, which had an increase in uptake of approximately 3-fold. Dimethyl-β cyclodextrin increased uptake most dramatically into the olfactory bulb. These results suggest that cyclodextrins can indeed enhance uptake of peptides by brain. More interestingly, they suggest that targeting of peptides to various brain regions may be possible by varying the cyclodextrin used.
Absorption of I-GALP after intranasal administration had a saturable component. In these experiments, no cyclodextrin was included in the intranasal administration. Uptake by whole brain of I-GALP was reduced approximately 50% when the I-GALP was coadministered with 1 µg of unlabeled GALP. Olfactory bulb showed an approximately 40% decrease, but this decrease did not reach statistical significance, probably because of the large variance in the data for this experiment. A decrease in I-GALP levels in carotid artery serum is consistent with entry into blood occurring by way of efflux from brain. The amount of GALP entering the brain after intranasal administration can be calculated by multiplying the dose of GALP by the %Inj/g for that brain region and then dividing the value by 100. By this formula, approximately 7.2 µg of GALP was taken up per gram of olfactory bulb and approximately 0.2 µg of GALP per gram of whole brain after the intranasal administration of 1 mg of GALP.
In conclusion, these results support the intranasal route as a viable way to administer GALP to the brain. The results also show that uptake by brain regions and distribution among peripheral tissues is very different for the i.n., i.c.v., and i.v. routes of administration. This, in turn, suggests that the relative mix of CNS and peripheral effects will differ drastically with these different routes of administration. Finally, the results suggest that targeting of various brain regions may be manipulated by the use of various cyclodextrins.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: GALP, galanin-like peptide; GALR, galanin receptor; BBB, blood-brain barrier; OB, olfactory bulb; i.n., intranasal; I-GALP, 131I-GALP; LR, lactated Ringer's solution; BSA, bovine serum albumin; WBr, whole brain; ant brain, anterior one third of the brain; %Inj/ml, percentage of the injected dose present in a milliliter of serum; %Inj/g, percentage of the injected dose taken up per gram of brain tissue; B-CD, dimethyl-β-cyclodextrin; A-CD, -cyclodextrin; CD, cyclodextrin; CSF, cerebrospinal fluid; TCA, trichloroacetic acid; ANOVA, analysis of variance; CNS, central nervous system.
Address correspondence to: Dr. William A. Banks, Geriatric Research, Education, and Clinical Center, Veterans Affairs Medical Center, John Cochran Division, 915 N. Grand Blvd., St. Louis, MO 63106. E-mail: bankswa{at}slu.edu
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