Introduction

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The 5-HT_1A receptor has been implicated in many important regulatory processes in animals and in the etiology and treatment of anxiety and depression. Pharmacotherapy mediated via the 5-HT_1A receptor is still in its infancy, and the possibility of developing new drug therapies based on this receptor is considerable.

The role of the 5-HT_1A receptor in regulating biochemical, neuroendocrine, and behavior processes has been elucidated mainly in animal studies, because the most selective compounds have only been available for preclinical studies. Of these, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) has been used most widely. It is a selective agonist for 5-HT_1A receptors, although it also has moderate affinity for the 5-HT₇ receptor (Plassat et al., 1993). Unfortunately, 8-OH-DPAT has poor oral activity in animals. Therefore, despite its interesting pharmacological profile, it has never been developed for use in humans.

8-OH-DPAT has a very wide range of effects in animals. It stimulates corticosterone release via the postsynaptic 5-HT_1A receptors (Lorens and Van de Kar, 1987; Przegaliñski et al., 1989) and inhibits serotonin synthesis and turnover via the somatodendritic 5-HT_1A autoreceptors (Hjorth and Magnusson, 1988). It also produces hypothermia (Hjorth, 1985; O'Connell et al., 1992) and a typical syndrome of behaviors, including flat body posture, forepaw treading, and lower lip retraction, which form part of the "serotonin-syndrome" (Tricklebank et al., 1984; Lucki, 1992). 8-OH-DPAT is also active in animal models of anxiety, although generally it does not have such a robust effect as benzodiazepines. It is also active in many screening tests for antidepressants in animals (Handley and McBlane, 1993).

Knowledge of the clinical functions of the 5-HT_1A receptor in psychiatric illness, in regulation of body temperature and hormonal control, and psychomotor performance, has come primarily from using substances from the class of azapirones. The most commonly used of these have been buspirone, ipsapirone, and gepirone. The only one that has received approval for medical use is buspirone. It has a documented anxiolytic effect in humans (Goldberg and Finnerty, 1979) and has also been shown to have antidepressant effects in double-blind studies (Feighner and Boyer, 1990). However, buspirone has a number of disadvantages as a tool for understanding 5-HT_1A function in humans. First, it has significant affinity for the dopamine D₂ receptor in which it acts as an antagonist. Second, it undergoes rapid first pass metabolism, giving it poor oral bioavailability. One of the major metabolites is 1-phenylpiperazine, which has significant affinity for -adrenergic receptors (Gower and Tricklebank, 1988). The relevance of this metabolite to the clinical effect is unknown. However, in some animal models, 1-phenylpiperazine can prevent the antidepressant-like effect of buspirone and related compounds (Fuller and Perry, 1989). Finally, buspirone has been found to act as a partial agonist at the 5-HT_1A receptor (Yocca et al., 1986). This means that under certain circumstances it may act as an agonist and others as an antagonist, depending upon the receptor reserve and serotoninergic tone. The other azapirones that have been tested in the clinic (i.e., ipsapirone, gepirone, and tandospirone) share some, but not all, of these properties. All are more or less rapidly metabolized, and none are selective for the 5-HT_1A receptor.

Among other 5-HT_1A receptor agonists flesinoxan has been reported to be an effective anxiolytic and antidepressant in phase II clinical trials (Grof et al., 1993; Bradford and Stevens, 1994). However, further clinical development of this compound appears to have been terminated recently (Levine and Potter, 1999).

(R)-3,4-Dihydro-N-isopropyl-3-(N-isopropyl-N-propylamino)-2H-1-benzopyran-5-carboxamide (NAE-086) (Fig. 1) is the result of a program of chemical synthesis based upon the aminotetralin and aminochroman structures. The objective was to produce a compound with pharmacological characteristics similar to those of 8-OH-DPAT but with adequate bioavailability and oral activity in animals, and eventually in humans, to allow development as a pharmacotherapy in psychiatric disorders (Hammarberg et al., 2000). Clinical data from the azapirones and flesinoxan together with data from animal models with 8-OH-DPAT suggest that such a compound should be therapeutically active in anxiety and depression in humans. The present paper describes the in vitro and in vivo pharmacological properties of NAE-086. We show that it is a very selective and efficacious 5-HT_1A receptor agonist and that it produces rapid tolerance to functional postsynaptic 5-HT_1A receptor responses but simultaneously sensitizes 5-HT₂ receptor responses.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   The chemical structures of NAE-086, the S-enantiomer NAE-084 and metabolites found in rat urine.

	Materials and Methods

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Compounds. The following radioactive compounds were used in the binding studies (the receptor and specific activity in parentheses): [³H]RX821002 (₂-adrenoceptor; 53.0 Ci/mmol), [³H]strychnine (strychnine sensitive glycine site; 23.5 Ci/mmol), [³H]DAMGO (µ-opioid; 59 Ci/mmol), [³H]DPDPE (-opioid; 33 Ci/mmol), [¹²⁵I]neuropeptide Y (neuropeptide Y; 2118 Ci/mmol) and [¹²⁵I]somatostatin (somatostatin; 1867 Ci/mmol) from Amersham Pharmacia Biotech UK, Ltd. (Little Chalfont, Buckinghamshire, UK); [³H]8-OH-DPAT (5-HT_1A; 135.5 and 162.9 Ci/mmol), [³H]5-HT (5-HT_1B and 5-HT₆; 23.4 and 28.2 Ci/mmol), [³H]ketanserin (5-HT_2A; 64.6 and 60.1 Ci/mmol), [³H]prazosin (₁-adrenoceptor; 25.0 and 72.2 Ci/mmol), [³H]rauwolscine (₂-adrenoceptor; 82.3 Ci/mmol), [³H]dihydroalprenolol (-adrenoceptor; 60.4 and 59.2 Ci/mmol), [³H]SCH23390 (dopamine D₁; 81.0 and 85.5 Ci/mmol), [³H]L-quinuclidinylbenzilate (muscarinic; 44.3 and 36.4 Ci/mmol), [³H]nicotine (nicotinic; 63 Ci/mmol), [³H]pyrilamine (histamine H₁; 31.2 Ci/mmol), [³H]tiotidine (histamine H₂; 83.7 Ci/mmol), [³⁵S]TBPS (GABA_A; 69.2 Ci/mmol), [³H]MK-801 (N-metyl-D-aspartate; 20.3 Ci/mmol), [³H]DL--amino-3-hydroxy-5-methylisoxazole-4-propionic acid (DL--amino-3-hydroxy-5-methylisoxazole-4-propionic acid; 53 Ci/mmol), [³H]flunitrazepam (benzodiazepine; 82.5 Ci/mmol), [³H]paroxetine (5-HT uptake site; 23.1 Ci/mmol), [¹²⁵I]galanin (galanin; 2200 Ci/mmol), [³H]mesulergine (5-HT_2C; 78 Ci/mmol), [³H]BRL 43694 (5-HT₃; 80 Ci/mmol), [³H]baclofen (GABA_B; 30.6 Ci/mmol), [³H]raclopride (dopamine D₂, D_2A, and D₃; 46.0 Ci/mmol), [³H]L-354,718 (CCK_A; 87 Ci/mmol) and [³H]L-365,260 (CCK_B; 73 Ci/mmol) were obtained from PerkinElmer Life Sciences (Boston, MA). NAE-086, [propyl-2,3-³H] (specific activity, 40.2 Ci/mmol) and NAE-086, [amido-¹⁴C] (specific activity, 52 mCi/mmol) were synthesized by Dr. Tom Werner, AstraZeneca R&D Södertälje (formerly Astra Arcus).

Cells. GH₄ZD10 (rat pituitary tumor) cells expressing the 5-HT_1A receptor and Ltk (mouse fibroblast) cells expressing human dopamine D_2A (long isoform) and D_2B (short isoform) were obtained from Dr. Olivier Civelli (Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, OR). Chinese hamster ovary cells expressing human D₃, rat 5-HT₆, and rat 5-HT₇ receptors were purchased from INSERM Institute (Paris, France).

Animals. Male Sprague-Dawley rats (ALAB or B&K strain) were supplied by B&K Universal (Sollentuna, Sweden) (formerly ALAB Laboratorietjänst). Dogs (beagles) for the in vivo metabolism studies were supplied by Vema Hund AB (Örbyhus, Sweden). The experiments were approved by Stockholm South ethical committee for experiments on laboratory animals.

In Vitro Receptor Binding Studies. Receptor binding assays for 5-HT_1A, bovine 5-HT_1B, 5-HT_2A, 5-HT₆, 5-HT₇, D₁, D₂, D_2A, D_2B, D₃, ₁, ₂, , muscarinic, nicotinic, histamine H₁ and H₂, GABA_A, glutamate and galanin receptors, and the serotonin uptake site were carried out under conditions described previously (Jackson et al., 1995; Johansson et al., 1997). Receptor binding assays for µ- and -opioid receptors were carried out as described by Magnan et al. (1982).

1

1

VIP-Stimulated Adenylyl Cyclase in GH₄ZD10 Cells. The GH₄ZD10 cells expressing 5-HT_1A receptors were cultured as described previously (Albert et al., 1990, Fowler et al., 1992; Johansson et al., 1997). The adenylyl cyclase assay used was based on the method of Dorflinger and Schonbrunn (1983) with some minor modifications (Fowler et al., 1992; Johansson et al., 1997).

In Vivo Metabolism of NAE-086. Urine was collected over the first 24 h from rats and dogs administered [¹⁴C]NAE-086 solution. The pH of the urine was adjusted to pH 12 with 1 M NaOH. The urine was extracted with 3 volumes of chloroform or a mixture of hexane/diethyl ether/butanol (70:30:5). The organic phase was collected and evaporated to dryness, after which the residue was dissolved in mobile phase before HPLC chromatography and detection. A YMC C18 ODS-A column (150 × 4.6 S-3 µm) was used for HPLC fractionation. The mobile phase consisted of 0.05 M phosphate buffer, pH 2.5, containing 0.6 mM octyl sulfate and 17% acetonitrile. The flow rate was 1 ml/min. Detection was by UV absorption at 205 nm using a Spectra 100 (Spectra-Physics, San Jose, CA) spectrophotometer. The radioactive peaks were detected by a FLO-ONE/ - detector (Radiometric; Canberra Instrument Co., Meriden, CT). The HPLC pump used was an LKB 2150. The eluted peaks were compared with those of synthetic reference compounds. A gas chromatography-mass spectrometry procedure was used for structural confirmation of the separated urinary metabolites.

5-HTP and DOPA Accumulation. The rats were administered with the test compound at the time noted before the intraperitoneal injection of 100 mg/kg NSD 1015. In some experiments, 6 mg/kg i.p. EEDQ was injected 24 h beforehand. The rats were killed with a guillotine 30 min after the NSD 1010 injections, the brains were removed rapidly, and the regions dissected were frozen immediately on dry ice. The samples were stored at 70°C until assayed.

5-HT and Dopamine Turnover. The test compound was administered at the time noted before the rats were decapitated. The brain regions to be analyzed were treated as described above.

Determination of 5-HTP, DOPA, 5-HT, Dopamine, and Their Metabolites. The concentrations of 5-HTP, DOPA, 5-HT, 5-HIAA, dopamine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid in various brain regions were determined by the use of high-performance liquid chromatography with electrochemical detection according to a modification of the method of Magnusson et al. (1980) as described previously (Johansson et al., 1997).

Serum Corticosterone. Rat serum corticosterone was determined as described by Kelder and Ross (1992). The rats were treated with saline injections once daily for 5 days before the experiment to reduce stress-induced corticosterone increase.

Flat Body Posture and Forepaw Treading. Flat body posture and forepaw treading were determined as described by Johansson et al. (1997). The intensity of the behavior was tested between 10 and 12 min after the administration of the test compound.

Temperature Measurements. Rectal temperature was measured as described by Johansson et al. (1997). Rectal temperature was recorded before the administration of the test compound and 30, 60, 90, and 120 min thereafter.

Cage-Leaving Response. The cage-leaving response in rats was determined as described by Rënyi et al. (1986). The number of rats leaving the cage and the time to leave the cage during the observation period 10 to 22 min after the test compound were recorded.

Wet-Dog Shake Response. Wet-dog shake response in rats was measured as described by Ross et al. (1992). The number of rats showing wet-dog shakes and the number of wet-dog shakes during the observation period 5 to 65 min after the challenge dose were recorded.

Salivation and Male to Male Mounting with Penile Erection. Salivation and male to male mounting with penile erection were observed during the cage-leaving test.

Statistics. The mean and S.E.M. were used as measures of central tendency and variation, respectively. Analysis of variance (ANOVA) was performed using SYSTAT 7.0: New Statistics (SPSS Inc., Chicago, IL), followed by a post hoc test as stated for each experiment. In some behavioral experiments Mann-Whitney U test was used. Medians ± quartiles were calculated according to Snedecor and Cohran (1967).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

In Vitro Experiments

Receptor Binding Profiles of NAE-086 and Its Metabolites. Both NAE-086 and the metabolite NAE-111 showed high affinity and selectivity for rat hippocampal 5-HT_1A receptors with K_i values of 4.5 ± 0.7 nM (n = 8) and 0.26 ± 0.03 nM (n = 3), respectively (Table 1). The S-enantiomer of NAE-086 (NAE-084) had more than 200 times less potency (K_i 1100 ± 200 nM). Other related compounds had low affinity for the 5-HT_1A receptor. Apart from an affinity of 240 nM at 5-HT₇ receptor the affinity of NAE-086 was less than 500 nM for all other 30 receptors studied (data not shown; see Materials and Methods for list of receptors tested).

Kinetics of [³H]NAE-086 Binding. The time course of the binding of [³H]NAE-086 to membranes of rat cerebral cortex including hippocampus at 22°C is shown in Fig. 2. The observed association rate constant k_obs obtained from the linear relationship was 0.0835 and the forward rate constant (k₊₁ = k_obs  k₁/[L]) for the [³H]NAE-086 binding at 22°C was 0.009 × 10⁹ M¹min¹. The dissociation rate constant (k₁), determined by addition of 10 µM 5-HT, was 0.0315 min¹. The dissociation was completed after 60 min with a half-time dissociation value (t_1/2) of 22 min (graphically determined). The calculated ratio of the rate constants (k₁/k₊₁) gave an estimate of the K_d value of 3.5 nM, which is in good agreement with the K_i value obtained using [³H]8-OH-DPAT, i.e., 4.5 ± 0.7 nM.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Association (A) and dissociation (B) of the binding of [3H]NAE-086 to membranes of rat cerebral cortex + hippocampus. Each value is the mean ± S.E.M. (vertical bars) of triplicate determination. A, membranes corresponding to 10 mg of original wet weight tissue were incubated with 5.8 nM [3H]NAE-086 for various times before collection and washing on Whatman GF/B filters by vacuum filtration. Nonspecific binding was determined in the presence of 10 µM 5-HT. Inset, graph of ln B60'/(B60'  B) versus incubation time, where B60' is the amount bound after 60 min and B that at the different times. The observed rate constant was 0.0835. B, membranes were incubated with [3H]NAE-086 for 60 min, whereafter the incubation was continued for different times in the presence of 10 µM 5-HT until they were collected by filtration. Inset, ln B/B0 versus incubation time where B is the amount bound at different times and B0 that at time 0.

Effects on VIP-Stimulated cAMP Production. 5-HT (1 µM), 8-OH-DPAT (1 µM), and buspirone (3 µM) reduced the cAMP response to VIP by 55, 50, and 20%, respectively (Table 2). These responses could be antagonized by 10 µM ()alprenolol. ()Alprenolol per se did not affect VIP-stimulated cAMP production. NAE-086 and its enantiomer, NAE-084, were tested concomitantly with the standard compounds. NAE-086 reduced the cAMP response concentration dependently, the maximum reduction (at 10 µM) being 79% of that found with 1 µM 5-HT (Fig. 3). The combination of 10 µM NAE-086 + 1 µM 5-HT produced a slightly lower inhibition of cAMP than that seen with 1 µM 5-HT alone. This suggests that the inhibition seen at 10 µM NAE-086 is close to the maximum obtainable for this compound. The concentration of NAE-086 producing half the inhibition observed at 10 µM was determined graphically from the mean data to be 220 nM. The inhibitory effect of 1 µM NAE-086 was antagonized by 1 to 10 µM ()alprenolol (Fig. 3, insert). NAE-084 (10 nM-10 µM concentration range) was without effect upon either VIP-stimulated cAMP production per se or upon the inhibitory effect of 1 µM 5-HT (data not shown). This indicates that over this concentration range, NAE-084 does not interact with the 5-HT_1A receptor-adenylyl cyclase complex.

View this table: [in this window] [in a new window]   TABLE 2 Effects of 5-HT, 8-OH-DPAT, buspirone and ()alprenolol on VIP-stimulated cAMP production in GH4ZD10 cells The results are means ± S.E.M., n = 4. The basal activity of the control samples was 440 ± 40 pmol of cAMP formed during the incubation/106 viable cells.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Dose-response curve of the inhibition by NAE-086 of VIP-stimulated cAMP production in GH4ZD10 cells expressing 5-HT1A receptors. The effect of NAE-086 in the presence of 1 µM 5-HT is also shown. Inset, dose-response curve of ()alprenolol (5-HT1A and -adrenergic receptor antagonist) to antagonize the effect of 1 µM NAE-086. Data are means ± S.E.M. of four experiments. The absolute activity of the control samples was 440 ± 40 pmol of cAMP formed during the incubation/106 viable cells.

In Vivo Experiments

Minimum effective doses of NAE-086 in various in vivo experiments are summarized in Table 3.

In Vivo Metabolism of NAE-086. A total of seven metabolites were identified in the urine of the rat and dog (Fig. 1). (R)-3,4-Dihydro-3-(N-ethyl-N-isopropylamino)-N-isopropyl-2H-1-benzopyran-5-carboxamide was the most predominant metabolite in rat urine, accounting for as much as 20% of the dose. (R)-3,4-Dihydro-N-isopropyl-3-(N-isopropylamino)-2H-1-benzopyran-5-carboxamide and (R)-3-amino-3,4-dihydro-2H-1-benzopyran-5-carboxamide were the most predominant urinary metabolites in dogs accounting for levels as high as 17 and 9%, respectively. NAE-111 levels amounted to about 3% of dose in rat urine, whereas only trace amounts were present in the urine of dogs.

5-HTP Accumulation. NAE-086 reduced dose dependently the accumulation of 5-HTP. The time course of this decrease in hypothalamus after s.c. (1 mg/kg) and p.o. (3 mg/kg) administration is shown in Fig. 4. The time courses in the other three brain regions examined were similar (data not shown). The maximal effect after s.c. administration was seen when NAE-086 was given 30 min before NSD 1015 and the rats were killed 30 min thereafter, whereas that after p.o. administration was seen when NAE-086 was given 1 h before NSD 1015. At the doses of NAE-086 used here, the maximal s.c. effect was about 65% of the total 5-HTP formation in control rats whereas the maximal effect after p.o. administration was about 50%. The duration of the effect was quite long-lasting, with significant effects 4 h after administration. The dose-response relationship was determined when NAE-086 was given s.c. 30 min before NSD 1015 (Fig. 5). This experiment also included rats pretreated with 6 mg/kg i.p. EEDQ 24 h beforehand. This compound irreversibly destroys many receptors, including 5-HT_1A receptors. Because of large receptor reserve of 5-HT_1A receptors in the raphe nuclei, by partially inactivating receptors, EEDQ shifted the dose-response curves to the right without changing the maximal response. The ratio between the ED₅₀ doses with and without EEDQ treatment (q) gives an estimation of the fraction receptors not inactivated by EEDQ and also gives an estimation of the intrinsic efficacy of the compound (Minneman and Abel, 1984; Cox et al., 1993). As shown in Table 4 the q values obtained for the different agonists suggest a reserve of 5-HT_1A receptors in the raphe nuclei of about 80% for NAE-086 and 8-OH-DPAT and 50 to 60% for buspirone.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Time courses of the inhibition of 5-HT synthesis in hypothalamus after 1 mg/kg s.c. or 3 mg/kg p.o. NAE-086. NAE-086 was administered at the time noted before the injection of NSD 1015 and the animals were decapitated 30 min thereafter. Each value is the mean of five rats ± S.E.M. (vertical bars). Statistical significance (*, P < 0.05) was calculated by Dunnett's t test after ANOVA. 5-HTP control value: 1.37 ± 0.05 nmol/g of tissue.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Dose-response curves of the inhibition of 5-HT synthesis by NAE-086 in four brain regions (hypothalamus, hippocampus, striatum, and frontal cortex). NAE-086 was injected 60 min before the injection of NSD 1015 and the animals were decapitated 30 min thereafter. Some rats were pretreated with 6 mg/kg s.c. EEDQ 24 h before the experiment (open symbols). Each value is the mean of five rats ± S.E.M. (vertical bars). Statistical significance (*, P < 0.05) was calculated by Dunnett's t test after ANOVA.

5-HT Turnover (5-HIAA/5-HT). After oral administration, NAE-086 reduced the 5-HIAA/5-HT ratio in the rat brain by about 40% in hypothalamus and frontal cortex but less in hippocampus and particularly in striatum (Fig. 6A). In this experiment the inhibition in frontal cortex was also significant at the lowest dose (0.1 mg/kg) examined (Fig. 6B). The low inhibition of the 5-HIAA/5-HT ratio in striatum was also observed in the rats treated with NSD 1015 after oral administration of NAE-086 (Fig. 6C). The corresponding effects of subcutaneous NAE-086, also in NSD 1015-treated rats, showed less pronounced inhibition in striatum than in the other brain regions, and the inhibition in frontal cortex was similar to that in hypothalamus (Fig. 6D). Subcutaneous administration of 8-OH-DPAT in NSD 1015-treated rats also produced less of an effect in striatum than other regions examined (Fig. 6E).

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Inhibition of the metabolism (turnover) of 5-HT expressed as the ratio 5-HIAA/5-HT in four different brain regions. A, time course of the effect of 3 mg/kg p.o. NAE-086. B, dose-response of the inhibition 60 min after orally administered NAE-086. C, dose-response of the inhibition of 5-HIAA/5-HT ratio in NSD 1015-treated rats (same experiment as that shown in Fig. 5). The rats were killed 90 min after orally administered NAE-086 (30 min after 100 mg/kg s.c. NSD 1015). D, dose-response of the inhibition by NAE-086 in NSD 1015-treated rats. The rats were killed 60 min after subcutaneously administered NAE-086. E, dose-response of the inhibition by 8-OH-DPAT in NSD 1015-treated rats, which were killed 60 min after subcutaneous administered 8-OH-DPAT. Upper curves show the time courses after 3 mg/kg p.o. NAE-086. Each value is the mean of five rats ± S.E.M. (vertical bars). Statistical significance (*, P < 0.05) was calculated by Dunnett's t test after ANOVA. Control values for 5-HIAA/5-HT in A (n = 5): hypothalamus, 0.64 ± 0.06; hippocampus, 0.87 ± 0.08; striatum, 1.01 ± 0.08; frontal cortex, 0.41 ± 0.04.

DOPA Accumulation. A significant increase in DOPA accumulation in hypothalamus was found 30 min after 3 mg/kg p.o. NAE-086 (128 ± 8% of controls, p < 0.05; Dunnett's t test). No significant increases were obtained in other regions.

Dopamine Turnover. The ratio of 3,4-dihydroxyphenylacetic acid/dopamine in striatum was increased significantly (153 ± 10%; p < 0.05; Dunnett's t test) 30 min after 3 mg/kg p.o., but the effect had already disappeared 30 min later.

Corticosterone Secretion. NAE-086 increased serum corticosterone in rats with a significant effect at 0.3 mg/kg s.c. and 1 mg/kg p.o. when determined 1 h after administration (Fig. 7A). This effect had disappeared 2 h after administration (Fig. 7B). The increase in corticosterone after a challenge dose (1 mg/kg s.c.) of NAE-086 was attenuated significantly 24 h after single doses of either 3 mg/kg s.c. or 10 mg/kg p.o. NAE-086 (Fig. 7C).

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Effect of NAE-086 on corticosterone secretion in Sprague-Dawley rats (ALAB strain). Each value is the mean ± S.E.M. from five (in B four) rats. *, p < 0.05 versus controls (Student-Newman-Keul t test after ANOVA. A, dose-response of the increase in serum corticosterone 60 min after subcutaneous or oral NAE-086. Control value: 23 ± 3 ng of corticosterone/ml. B, time course of the rat serum corticosterone after 1 mg/kg s.c. or 3 mg/kg p.o. NAE-086. Control value: 174 ± 45 ng corticosterone/ml. C, attenuation of the increase in serum corticosterone 24 h after a single administration of NAE-086. Groups of five rats were administered subcutaneously or orally with saline or three different doses NAE-086 24 h before a challenge dose of 1 mg/kg s.c. NAE-086 and the rats were killed 60 min later. Control values: saline + saline, 64 ± 8 ng of corticosterone/ml; saline + NAE-086, 620 ± 43 ng/ml.

Flat Body Posture and Forepaw Treading. NAE-086 induced the 5-HT_1A receptor-mediated behavior syndrome with minimum effective dose of 0.3 mg/kg s.c. and 10 mg/kg p.o. (Table 3). The corresponding effect of 8-OH-DPAT was obtained at 0.3 mg/kg s.c. and >5 mg/kg p.o.

Hypothermia. NAE-086 significantly and dose dependently decreased rectal temperature in rats at 0.3 and 1 mg/kg s.c. and 1 to 10 mg/kg p.o (Fig. 8, A and B). NAE-111 also potently reduced rectal temperature also at the lowest dose tested, 0.03 mg/kg s.c., and showed a minimum effective oral dose of 1 mg/kg (Fig. 8, C and D).

View larger version (31K): [in this window] [in a new window]   Fig. 8.   Hypothermic effects of NAE-086 (A and B) or NAE-111 (C and D) in rats. Rectal temperature was measured before (0) and different times after the administration of the test compound. Doses (mg/kg) are shown in the figures. A, subcutaneous NAE-086. B, oral NAE-086. C, subcutaneous NAE-111. D, oral NAE-111. Each value is the mean ± S.E.M. of five to six rats. Statistical significance (*, p < 0.05) was calculated by Dunnett's t test after ANOVA.

Cage-Leaving Response. Acute administration of NAE-086 inhibited the cage-leaving response at a minimum effective dose of 1 mg/kg s.c. and 3 mg/kg p.o. (Table 3).

Attenuation of 5-HT_1A Receptor Responses. A single administration of NAE-086 to ALAB Sprague-Dawley rats attenuated the effect of a challenge dose of 8-OH-DPAT or NAE-086 given 24 h later in the following tests: corticosterone secretion (Fig. 6C), hypothermia (Table 5), and the cage-leaving response (Table 6). B&K Sprague-Dawley rats were less sensitive in developing tolerance to the 8-OH-DPAT-induced 5-HT_1A syndrome (flat body posture and forepaw treading) and the inhibition of cage-leaving response, but these parameters were attenuated markedly 24 h after the last administration of 3 weeks treatment with 1 mg/kg s.c. or 3 mg/kg p.o. NAE-086 twice daily (Fig. 9; Table 7).

View larger version (24K): [in this window] [in a new window]   Fig. 9.   Effects of 3-week treatment of rats with NAE-086 or 8-OH-DPAT on the behavioral changes flat body posture and forepaw treading induced by challenge doses of 8-OH-DPAT 24 h after the last repeated administration. The intensity of the behaviors was scored (0-4) between 10 and 12 min after the injection of 8-OH-DPAT. Values are mean scores from groups of 7 to 10 rats.

Wet-Dog Shake Response. 5-HT_1A receptor agonists do not induce wet-dog shakes because these are mediated by 5-HT₂ receptors. However, as reported previously (Ross et al., 1992) the number of spontaneous wet-dog shakes was increased significantly 24 h after a single administration of 8-OH-DPAT to ALAB Sprague-Dawley rats. As shown in Table 6, NAE-086 at 10 mg/kg s.c. or p.o., like buspirone and other 5-HT_1A receptor agonists, significantly increased spontaneous wet-dog shakes in the ALAB rat strain when observed 24 h later. In the B&K rat strain neither 0.1 mg/kg s.c. 8-OH-DPAT nor 1 mg/kg s.c. NAE-086 increased the number of spontaneous wet-dog shakes or those induced by 0.3 mg/kg s.c. DOI when tested 24 h later (Table 8). Repeated treatment of rats twice daily for 3 weeks with 8-OH-DPAT and NAE-086 did, however, double the number of DOI-induced wet-dog shakes when challenged 24 h after the last administration in this strain (Table 8).

Salivation and Male-to-Male Mounting with Penile Erection. Acute treatment with doses of up to 30 mg/kg s.c. 8-OH-DPAT did not induce salivation or male-to-male mounting with penile erection in B&K Sprague-Dawley rats (Table 7). However, 24 h after the last administration of 3 weeks, twice daily, treatment with 0.1 mg/kg s.c. 8-OH-DPAT-treatment, a challenge dose of 1 mg/kg s.c. 8-OH-DPAT caused pronounced salivation, and male-to-male mounting with penile erection was observed during a period 3 to 12 min after injection. Similar effects were induced after 3 weeks treatment twice daily with 1 mg/kg s.c. or 3 mg/kg p.o. NAE-086.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In Vitro Effects. The in vitro receptor binding profile of NAE-086 indicates that this ligand is one of the most selective 5-HT_1A receptor agonists so far reported. The only other receptor of the 32 examined for which NAE-086 had some affinity was the 5-HT₇ receptor, and this was 50 times less than for the 5-HT_1A receptor. The S-enantiomer of NAE-086 (NAE-084) has a much lower affinity for the 5-HT_1A receptor, demonstrating a greater degree of stereoselectivity for this receptor than previously reported for 8-OH-DPAT.

In Vivo Biochemical Effects. The results obtained with the 5-HTP accumulation technique confirm that NAE-086 is a potent 5-HT_1A receptor agonist under in vivo conditions after both subcutaneous and oral administration. They also show that the efficacy of NAE-086 is similar to that of racemic 8-OH-DPAT. These results complement the data from hippocampal membranes, to show that the agonist effects of NAE-086 are also evident at the presynaptic, somatodendritic 5-HT_1A autoreceptors. The rapid onset of the effect after s.c. injection with maximal effect within 30 min indicates that it is mainly NAE-086 itself and not a metabolite that exerts the effect at the receptor under these conditions. After oral administration it is more likely that the compound is subjected to first-pass metabolism and that the active metabolite NAE-111 contributes to the overall pharmacological effect. The rather flat dose-response curves after oral administration shown in Fig. 5 may reflect this.

Behavioral Effects. Many of the 5-HT_1A receptor-mediated responses are originated postsynaptically, e.g., the behavioral syndrome, corticosterone secretion, and probably also the hypothermia (Lucki, 1992). We have observed that the same rat strain from different breeders differs in sensitivity for 5-HT_1A receptor-mediated effects, both in the potency to activate the receptors and to desensitize the receptors upon repeated treatment (Kelder and Ross, 2001). Thus, the ALAB Sprague-Dawley strain from B&K Universal and the Sprague-Dawley strain from Møllegaard (Vejle, Denmark) had high sensitivity for 8-OH-DPAT and the responses were attenuated strongly after a single dose (Larsson et al., 1990; Kelder and Ross, 1992; Rënyi et al., 1992; Ross et al., 1992). Other Sprague-Dawley rat strains, e.g., from B&K and from Charles River (Uppsala, Sweden), were less sensitive for this class of compounds and require repeated treatment to show attenuation of the receptor responses (Kelder and Ross, 2001). These discrepancies make comparisons between laboratories difficult and even comparisons between data obtained at different times in the same laboratory. Regardless of these differences, it is well established that 5-HT_1A receptor agonists cause a development of tolerance upon repeated treatment (for review, see De Vry, 1995). As shown in the present study NAE-086, like other 5-HT_1A receptor agonists, desensitized many 5-HT_1A receptor responses. Interestingly, however, it was found that, simultaneously with the development of the tolerance to the 5-HT_1A receptors, sensitization occurred to the 5-HT₂ receptor agonist DOI. Thus, in one experiment a single administration of NAE-086, like 8-OH-DPAT, induced an increase in spontaneous wet-dog shakes, a 5-HT_2A-mediated behavior (Schreiber et al., 1995), 24 h later. In another experiment, with less sensitive rats, 3 weeks of treatment with NAE-086 or 8-OH-DPAT increased the wet-dog shakes induced by a challenge dose of DOI when tested 24 h after the last administration of the 5-HT_1A agonist. This sensitization of the 5-HT_2A receptor response is probably a direct consequence of the tolerance toward the 5-HT_1A receptor responses, reflecting the balance between 5-HT_1A and 5-HT₂ receptor systems (Rënyi, 1991; Schreiber et al., 1995). The observations of salivation and male-to-male mounting with penile erection after a high challenge dose (1 mg/kg s.c.) of 8-OH-DPAT in rats that had been treated repeatedly with NAE-086 or 8-OH-DPAT are interesting and do not seem to have been reported previously. Although not further analyzed in the present study, these behavior changes indicate a strong sensitization of serotoninergic systems and may also involve other neuron systems, e.g., dopaminergic and cholinergic systems.