Nasal administration of a cognition enhancer provides improved bioavailability but not enhanced brain delivery

Nasal Administration of a Cognition Enhancer Provides Improved Bioavailability but Not Enhanced Brain Delivery MUNIRA. HUSSAIN,DARIELRAKESTRAW,S U S A N ROWE, AND

BRUCE J. AUNGST'

Received Au ust 25, 1989, from the DuPont Company, Medical Products Department, P.O. Box 80400, Wilmingfon, h E 19880-0400. Accepted for publication November 14, 1989. Abstract 0 Compound 1 [3,3-bis(4-pyridyImethyI)-l-phenylindolinQ-

one] is an experimental cognition-enhancingdrug now baing developed for cognitive disorders. Oral bioavailability of 1 in rats was 50%. Brain levels of total radioactivity were measured after iv and nasal doses of radiolabeled 1. The ratio of AUCbrain:AUCplasma was the same by both routes, so nasal dosing did not enhance brain delivery. This is in contrast to other reports of large molecular weight substances and metals gaining direct access to the brain through the nasal epithelium.

Nasal administration c a n improve the systemic bioavailability of drugs which undergo first-pass metabolism after oral dosing, and has frequently been used experimentally for t h i s purpose.' It has also been reported that nasal administration m a y provide access t o the brain through areas unprotected by the blood-brain barrier.2~3We were interested in seeing whether these advantages would apply to compound 1 [3,3-bis(4-pyridylmethyl)-l-phenylindolin-2-onel, a developmental cognition enhancer which is subject t o oral first-pass metabolism and for which the brain is the target organ. The structure o f t h i s compound is shown below ([l4C11 was labeled i n the position indicated by the solid circle). Compound 1has been shown to enhance acetylcholine release in rat brains in vitro and in vivo.4 Initial studies in humans using EEG b r a i n mapping suggested that 1 has v i g i l a n c e - i m p r o v i n g properties.5

Experimental Section Materials-Compound 1 and 14C-labeled 1 ([l4C11) were from Du Pont Pharmaceuticals and DuPont NEN Research Products, respectively. "C-labeled 1 was supplied as the free base having a specific activity of 21.96 mCi/mmol. It was 99% pure by TLC. Propranolol hydrochloride was obtained from Sigma Chemical. Bioavailability Studie-Male Lewis rats weighing -300 g were used. All rats were anesthetized with 50 mgkg pentobarbital (Nembutal) ip. Compound 1 was administered intravenously, orally, and nasally to estimate oral and nasal bioavailability. For iv administration, a dose of 1 .2HC1 equivalent to 2 mgkg was administered by cardiac puncture in a volume of 1 m u g . "he oral dose was 20 mgkg in a volume of 1m u g , and it was administered by gavage. The nasal dose was administered to rats afler surgical isolation of the nasal cavity. The trachea was cannulated to allow free breathing, and a closed tube was inserted anteriorly through the esophagus to block the posterior part of the nasal cavity. The incisive ducts were closed with an adhesive to prevent drainage into the mouth. The dose was administered through the nares with a microliter syringe. The dose of 1 . HCI was equivalent to 4 mgkg of the base, and the dosing volume was 0.25 m u g , administered through both nares. Serial blood samples (-0.5 mL) were collected into heparinized test tubes after cutting the tip of the tail. Plasma was separated and frozen until analyzed for 1. Plasma 1 concentrations were determined by HPLC after solvent extraction. First, 0.2 mL of plasma, 0.1 mL of internal standard solution (0.15 mg propranolol HCl/mL water), 0.1 mL of sodium carbonate solution (3.5% in water), and 5 mL of ethyl acetate were added to test tubes. The tubes were vortexed for 5 min and, after 0022-3549/90/0900-0771$0 1 .OO/O 0 7990,American Pharmaceutical Association

centrifugation, the organic layer was transferred to conical centrifuge tubes to which 0.25 mL of 0.1 M HCl was added. The tubes were vortexed for 1 min and, after centrifugation, the organic phase was removed. Aliquots of the acid were injected onto the HPLC. Separation of 1 and the internal standard was achieved at ambient temperature on a 4.6 x 250-mm cyano column (Zorbax CN, DuPont) attached to a guard column packed with cyano packing. The proportion of components in the mobile phase was 620 mL of 0.1 M acetate buffer at pH 3.5,480mL of acetonitrile, 5 mL of tetrahydrofuran, and 0.75 g of sodium heptanesulfonate. At a flow rate of 1.75 mumin, the retention times of 1 and the internal standard were 7.4 and 5.5 min, respectively. Detection was by UV absorbance a t 252 nm. The detection limit was 0.12 pg/mL plasma. The area under the plasma 1 concentration versus time curve (AUC) from 0 to 3 h was calculated for each rat. Systemic bioavailability after oral and nasal doses was calculated for each rat as the ratio of AUC,,,, or n,aal:AUCi,, using the average AUC,, and correcting for the differences in dose. Values are expressed as percent ofdose. Brain Uptake Study-Male Sprague-Dawley rats (Charles River) were anesthetized with pentobarbital and dosed with 2 mglkg of [14C]1intravenously or nasally. The dosing solution was prepared by dissolving 14 mg of 1 and 2 mg of [l4C11per milliliter of 0.1 M HCl. Intravenous dosing was through a jugular vein cannula and nasal dosing was as described above, except that the dosing volume was 0.125 mLkg. Animals were decapitated 2, 5, 10, 20, or 30 min after dosing and the draining blood was collected. Brains were removed, divided for duplicate assays, and oxidized in a Packard Oxidizer. Total radioactivity in plasma and brain specimens was determined by scintillation counting.

Results The plasma concentration versus time profiles for iv, oral, and nasal delivery of 1in rats are illustrated in Figure 1.Oral bioavailability averaged 9.1%of the dose (Table I). Additional studies not reported here showed that first-pass metabolism was primarily responsible for the loss of oral bioavailability. Nasal administration resulted in markedly improved bioavailability (Table I). Nasal absorption was rapid, as evidenced by the rapid attainment of maximum plasma concentrations (within 10 min). Results of the b r a i n u p t a k e study using [14C31are summarized in Table 11. Compound 1 e n t r y into the brain was rapid after i v o r n a s a l dosing. M a x i m u m brain and plasma concentrations were observed 2 min after iv dosing and 5 m i n after

1

Journal of Pharmaceutical Sciences I 771 Vol. 79,No. 9,September 7990

Table &Average (SEM) Braln and Plasma Concentratlons of ['4C]1 (Total Radloactlvlty) In Rats after Intravenous or Nasal Doslng ~~

Administration Route Intravenous

Sample Time, min 2 5 10 20 30 A'JCz-30rnIn

Nasal

2

5 10 20 30 A'JCzaomm

0.05 O.'I I

2

1

0

3

HOURS Figure 1-Plasma

concentrations (mean -e SEM) of 1 in rats after

2-mg/kg iv (O), 20-mg/kg oral (m), or 4-mg/kg nasal (A)doses. Table I-Oral and Nasal Bioavallablllty (F) of 1 In Rats Dose

intravenous (2 mg/kg) Oral (20 mg/kg) Nasal (4 mg/kg)

AUCSah, pg h mL-' 1.26 2 0.09 1.15 f 0.24 1.34 f 0.14

F, YO of Dose

N

100 9.1 t 1.9 53.0 -t 5.5

13 9 5

nasal dosing. Brain-to-plasma concentration ratios were fairly time independent, reflecting what appears to be rapid equilibrium. The exception to this trend was that 2 min after iv dosing, the brain-to-plasma concentration ratio was unexpectedly high. The area under the average brain or plasma concentration versus time curve (AUC) from 2 to 30 min was calculated. The brain-to-plasma AUC ratio represents a time-average partitioning ratio. This ratio was 0.30 for both intravenously and nasally administered 1. Brain uptake was independent of the route of administration.

Discussion The target organ for this cognition enhancer is the brain. One objective of these experiments was to determine whether nasal dosing could increase delivery to the brain. There have been several reports suggestingthat this might be possible. Gopinath et a1.2 showed that colloidal gold particlea deposited on the olfactory epithelium of monkeys by nasal spray were absorbed by the olfactory neurons and migrated as far as the fila olfactoria. 772 / Journal of Pharmaceutical Sciences Vol. 79, No. 9, September 7990

Brain,

Plasma,

ng4.l

ng/mL

Brain: Plasma

2593 (1 059) 1788 (273) 1.28 (0.31) 400 (1 3) 1194 (52) 0.34 (0.01) 216 (21) 1291 (25) 0.17 (0.02) 155 (10) 986 (51) 0.16 (0.01) 149 (21) 830 (74) 0.18 (0.01) 9405 31150 0.30 266 (13) 432 (56) 314 (46) 21 0 (24) 135 (29) 7257

726 (55) 0.37 (0.02) 1090 (120) 0.43 (0.09) 866 (91) 0.36 (0.02) 925 (30) 0.23 (0.03) 666 (97) 0.20 (0.01) 24524 0.30

Pinocytotic vesicles were observed in the olfactory neurons. Similarly, horseradish peroxide administered intranasally to mice, rats, and monkeys was absorbed through the olfactory epithelium and entered the olfactory bulbs.3 The mechanism of epithelial absorption was through the intercellular spaces. ARer chronic nasal exposure of rabbits to various aluminum salts, granulomas (apparentlyharboring aluminum)were identified in the cerebral cortex.6 Bradbury et al.7 showed that radiolabeled albumin injeded into the cerebral ventricles or caudate nucleus of rabbits could pass via the subarachnoid space of the olfactory lobes and olfactory neurons, through the dura and bone to the submucous space of the nasal epithelium. Nasally administered pendorphin was more effedive in elevating serum prolactin levels in monkeys than equal doses administered intravenous1y.s However, in a study by Anand Kumar et al.,9 nasally administered progesterone provided complete bioavailability, but cerebrospinal fluid:plasma ratios of progesterone concentrations were similar after iv and nasal doses. Our results are similar to those with progesterone, in that brain-to-blood ratios of 1 concentrations were the same following nasal and iv administration. This suggests that a direct pathway from the nasal epithelium to the brain may be significant only for poorly absorbed solutes like the proteins, peptides, and metals for which it has been described. Well-absorbed solutes, including 1, are apparently cleared rapidly to the systemic circulation and any neuronal absorption or absorption into the subarachnoid space is relatively slow and insignificant. The advantage of administering 1 nasally is that bioavailability was fivefold greater than after oral doses. Entry of the compound into the brain aRer iv and nasal delivery was demonstrated.

References and Notes 1. Chien, Y. W.; Su, K. S. E.; Chan S F. Nasal Systemic Drug Delivery; Marcel Dekker; New Yo%, i989 2. Gopinath, P. G.; Go inath, G.; Anand Kumar, T. C. Curr. Ther. Res. 1978,23,59d07. 3. Balin, B.J.; Broadwell, R. D.; Salcman, M; El-Kalliny, M. J . Comp. Neurol. 1986,251,260-280. 4. Nickolson, V .J.; Tam, S. W.; Myers, M. J.; Cook, L. FASEB J . 1989,3,A931. 5. Saletu, B.; Darragh, A.; Salmon, P.; Coen, R. Br. J . Clin. P h r mc01. 1989,28,1-16. 6. Perl. D. P.: Good. P. F. Lancet 1987,1, 1028. 7. Bradbury,'M. W..B.;Cserr, H. F.; Westrop, R. J. Am. J . Physiol. 1981,240,F329-F336. 8. Rao, A. J.; Moudal, N. R.; Li, C. H. Znt. J . Peptide Protein Res. 1986,28,546-548. 9. Anand Kumar, T. C.; David, G. F. X.; Sankaranarayanan, A.; Puri, V.; Sundram, K. R. Proc. Nut. Acad. Sci. 1982, 79,41854189.