Introduction

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Corticotropin-releasing factor (CRF¹) has been shown to play an important role in the behavioral and physiological response of the body to stress. In humans, CRF and CRF receptor disorders occur in some psychiatric diseases, such as major depression and anxiety (Owens and Nemeroff, 1991; Chalmers et al., 1996). Intracerebroventricular administration of CRF to rats produces similar effects to those observed in animals exposed to stress and are blocked by -helical CRF_9-41, a peptidic CRF receptor antagonist, suggesting that CRF receptors antagonists may be a useful medication for the treatment of mental illnesses, such as major depression or anxiety disorders (Owens and Nemeroff, 1991; McCarthy et al., 1999). Since peptides have little chance to penetrate the blood-brain barrier, attention has been focused on nonpeptidic compounds like CP-154,526 (Fig. 1), first described by Schulz et al. (1996). They showed that CP-154,526 has a high affinity for the CRF₁ receptors, which are responsible for the neuroendocrine regulation of the pituitary and for the cognitive and sensory functions, but not for the CRF₂ receptors, which regulate the neuroendocrine, autonomic, and behavioral actions of brain CRF (Lehnert et al., 1998). Schulz and coworkers (1996) also demonstrated that CP-154,526 blocks the CRF-induced adrenocorticotropin secretion in rats after s.c. application in a dose-dependent way. The compound also displayed antidepressant-like and anxiolytic effects in different in vivo experiments after i.p. or p.o. application (Lundkvist et al., 1996; Chen et al., 1997; Mansbach et al., 1997); however, Griebel et al. (1998) compared the behavioral profile of CP-154,526 with buspirone and diazepam in different anxiety models and found little anxiolytic activity. On the other hand, the compound attenuated the stress-induced reinstatement of drug seeking in rats (Shaham et al., 1998), and defensive withdrawal behavior of rats was decreased after 10 days of chronic treatment (Arborelius et al., 2000).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Structure of CP-154,526.

All these studies suggest that CP-154,526 crosses the blood-brain barrier, but there is no direct evidence for this. In this study, the pharmacokinetic profile of CP-154,526 in brain and plasma and its brain uptake index (BUI) were determined by radioactivity measurement and electrospray high-performance liquid chromatography-mass spectrometry (LC/MS).

	Materials and Methods

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Test Compound. The first part of this study was performed with [³H]CP-154,526 and the second part with nonlabeled substance. The labeling was carried out by the Isotope Laboratories of Novartis Pharma (Basel, Switzerland). Its purity was checked by HPLC. UV and radioactivity evaluation of the chromatograms showed a similar chemical purity as that of the reference standard and a radiochemical purity of >98%. The labeled batch used in this study had a specific radioactivity of 4096 GBq/mmol.

Pharmacokinetics. Oral plasma pharmacokinetic studies were performed with three male Wistar rats (275-310 g; Biological Research Laboratories, Füllinsdorf, Switzerland). The day before dosing, the rats underwent surgical implantation of an indwelling cannula from the right femoral artery to the back of the neck. The animals were individually housed in metabolism cages and were fasted overnight before administrations. The animals were weighed before treatment, and the dosis was calculated for each rat. For the oral dose (13.7 µmol/kg or 800 µCi/kg), [³H]CP-154,526 was dissolved in Placebo G drink solution (Sandoz Ltd., Basel, Switzerland) administered by gastric intubation. For the i.v. dose (2.7 µmol/kg or 1000 µCi/kg), [³H]CP-154,526 was dissolved in a mixture of ethanol (Merck, Darmstadt, Germany)/polyethylenglycol 200 (Merck)/glucose 5% (10:50:40, v/v/v) and administered into the surgically exposed femoral vein in the same animals as those used for the p.o. study after a 1-week interval. Blood samples were taken up to 48 h postdose from the cannulated femoral.

BUI. The unidirectional influx for [³H]CP-154,526 was measured by the brain-sampling single-injection technique (Oldendorf, 1970) in adult male Wistar rats (~220 g) under anesthesia [ketamine, 130 mg/kg i.m. (Graeub, Bern, Switzerland); xylazine, 1.3 mg/kg i.m. (Bayer, Leverkusen, Germany)]. A bolus of ~200 µl of either 0.01 M HEPES-buffered Ringer's solution, pH 7.4, or rat plasma was rapidly injected into the common carotid artery. The bolus contained [³H]CP-154,526 at a concentration of 84.9 or 16.4 nmol/ml (9 µCi/ml) together with [¹⁴C]butanol (1 µCi/ml; PerkinElmer Life Sciences, Boston MA), the amount of ethanol in the injectant was 0.7 to 1.2% (v/v). The animals were decapitated 5 s after the injection. Samples of the injection solution and the brain hemisphere ipsilateral to the injection side were solubilized in 2 ml of Soluene-350 (Packard, Meriden CT) at room temperature the night before double-isotope liquid scintillation counting. The percentage BUI was calculated as 100 · (³H/¹⁴C dpm)_brain/(³H/¹⁴C dpm)_injectant. The brain extraction ratio E was calculated from E = BUI · 0.73, where 0.73 represents the brain extraction ratio of [¹⁴C]butanol (Pardridge et al., 1985).

Sample Analysis. The concentration of [³H]CP-154,526 in blood was determined by LC-reversed isotope dilution. The procedure involved the addition of 13.7 nmol of nonradiolabeled CP-154,526 to each blood sample as an internal standard. After adding 1 ml of water, 100 µl of buffer, pH 9 (concentrated 5 times; Merck) and 4 ml of diethyl ether (Merck), the samples were shaken for 30 min and centrifuged (6000g, 5 min, 20°C). The supernatant was evaporated in a vacuum centrifuge (Univapo 150H; Zivy, Oberwil, Switzerland). The residue was reconstituted in 250 µl of mobile phase/water and centrifuged (3000g, 60 s). The supernatant (200 µl) was analyzed by HPLC (MT2; Kontron Instruments, Zürich, Switzerland) on a Waters Symmetry Shield RP-8 column (5 µm, 150 × 3.9 mm, 60°C; Milford MA) with 0.1% tetramethylammonium hydrogen sulfate/acetonitrile (60:40, v/v; Merck) as mobile phase. The flow rate was 1.2 ml/min; the effluent was monitored at 295 nm. The peak corresponding to the unchanged [³H]CP-154,526 was collected in a polyethylene vial by a fraction collector (SuperFrac; Amersham Biosciences AB, Uppsala, Sweden) and subjected to radioactivity determination. The concentration of [¹⁴C]CP-154,526 in each sample was calculated from the ratio of the amount of radioactivity in the eluate fraction corresponding to CP-154,526 and the area of the ultraviolet absorbance of the nonradiolabeled CP-154,526 used as an internal standard. Recoveries averaged 88 ± 6% for blood and 50 ± 10% for brain. The limit of quantification was dependent of the specific radioactivity (i.e., 1.8 and 0.3 pmol/ml after oral and i.v. administration, respectively).

Data Analysis. The peeling method was applied to describe the data by a biexponential model characterized by C = C₁ · e^₁t + C₂ · e^₂t. The initial estimates of C₁, ₁, C₂, ₂ were taken to generate the best fit using the computer software ELSFIT (Sheiner, 1981). The half-lives were calculated as t_1/2I = ln 2/_i. Areas under the curve (AUC) and areas under the first-moment curve were calculated by the trapezoidal rule and extrapolated to infinite time. Total clearance (CL) was calculated as dose/AUC_iv. The volume of distribution at steady state was calculated as V_SS = MRT · CL, where MRT is the mean residence time, calculated as areas under the first-moment curve/AUC.

	Results

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Based on the dose-normalized AUC ratios, an average bioavailability of 27 ± 6% was estimated. CP-154,526 displayed a large volume of distribution (V_SS = 105 l/kg), and a systemic CL of 36 ml/min/kg. After i.v. bolus, the concentration of CP-154,526 in blood declined biphasically with a first half-life of 0.9 h and a terminal half-life of 51 h (Table 1; Fig. 2).

View larger version (8K): [in this window] [in a new window]   Fig. 2.   Blood CP-154,526 concentrations after oral (13.7 µmol/kg) and intravenous (2.7 µmol/kg) administration (individual values and mean-fitted line).

The unidirectional brain extraction of [³H]CP-154,526 was 27 ± 6 and 9 ± 2% after the intracarotid injection of Ringer's buffer containing 0.08 or 16.4 nmol/ml of the compound, respectively. At both concentrations, the brain extraction was strongly decreased when the compound was dissolved in rat plasma to the low, but significant, values of 3 to 4%.

Brain pharmacokinetic was studied by comparing plasma levels of CP-154,526 with the concentration in five brain tissues, namely the cortex, hypothalamus, hippocampus, striatum, and cerebellum. After i.v. application, brain maximal concentrations were reached after ca. 20 min, and no significant differences were observed between the five studied brain tissues, except for the hypothalamus, which exhibited slightly lower concentrations, and for the cerebellum, which showed higher concentrations until 20 min post-treatment (Table 2; Fig. 3A). After i.v. application, the concentration ratio of brain part/plasma reached the maximum of about 2.5 after 1 to 2 h and then slowly decreased in all brain tissues, except again for hypothalamus, where the ratio never exceeded 1 (Fig. 4).

View larger version (10K): [in this window] [in a new window]   Fig. 3.   A, the CP-154,526 concentration in plasma and cortex after i.v. injection (10 µmol/kg); B, the CP-154,526 concentration in plasma and cortex after p.o. application (30 µmol/kg).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   The concentration ratios of brain part/plasma of CP-154,526 after i.v. (10 µmol/kg) (A) and p.o. (30 µmol/kg) (B) application. , cortex; , striatum; , cerebellum; , hippocampus; , hypothalamus.

After p.o. application, maximal plasma concentration was reached after ca. 30 min. Brain concentrations increased rapidly to reach maximal concentration after about 1 h and remained stable until 2 h post-treatment (Fig. 3B). No significant differences could be observed between the brain regions (Table 3). No differences in the ratios of brain tissues/plasma were noted between the brain tissues after p.o. application.

	Discussion

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CP-154,526 displayed a high tissue distribution, revealed by the large volume of distribution (105 l/kg). In accordance with this, an extensive brain distribution with high brain/blood concentrations ratios in both i.v. and p.o. applications could be demonstrated. The brain extraction ratios (9 to 27%) indicate a large and saturable brain penetration of CP-154,526, and the decrease of these ratios to 3 to 4% when using rat plasma as vehicle indicates high plasma protein binding. Since a fairly high systemic clearance (CL = 36 ml/min/kg) is observed, the slow elimination (t_1/2 = 51 h) may be attributed to the extensive tissue distribution.

CP-154,526 showed good penetration in the different brain tissues, with a maximum already reached after 20 min in the i.v. study. The brain/plasma ratios went up to 2.5 within 1 to 2 h and then slowly decreased, except in the hypothalamus, where the ratio never exceeded 1 and remained stable at this level until 4 h post-treatment. Several hypotheses can be formulated to explain this phenomenon. The blood perfusion is known to be different between the brain regions and, therefore, could influence the penetration of the substance into the brain. Since the transport system is saturable, it can also be supposed that saturation occurred faster in the hypothalamus than in the other brain tissues. Myelin is also distributed differently within the brain and, therefore, increases the lipophilia of the brain regions where it is present in higher concentrations. This means that a lipophilic substance will accumulate more and stay longer in the tissues with high amounts of myelin.

The rat hypothalamus is known to have a lower density of CRF₁ receptors in comparison with other brain tissues, like the brain cortex, whereas the cerebellar cortex displays a higher density of CRF₁ receptors (Chalmers et al., 1995, 1996). It is not clear whether the concentrations of CP-154,526 are a reflection of the receptor's densities, but since CP-154,526 is a highly specific CRF₁ receptor antagonist (Schulz et al., 1996), the possible consequences of the lower hypothalamus and higher cerebellum concentrations of CP-154,526 on its pharmacological activity have to be examined.

In the p.o. study, the concentration ratios still increased 8 h after application (Fig. 4), but no significant differences within the brain regions were observed. Differences between brain tissues seen only in the i.v. trial, where the concentrations are about 10-fold higher than in the p.o. experiment, suggest that a saturation occurs at high concentrations.

In summary, CP-154,526 has been proven to cross the blood-brain barrier in a saturable way well. Correlative studies between behavioral and brain concentrations should be undertaken to determine the minimal brain concentration required for activity, especially for p.o. studies, where the oral bioavailability of 27% can lead to subtherapeutic concentrations in the brain if not taken into account.