Pharmacokinetics and bioavailability after intramuscular injection of the 5-HT1A serotonin agonist R-8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) in domestic goats (Capra aegagrus hircus).

To determine the bioavailability and pharmacokinetic properties of the serotonin 5-HT1A receptor agonist R-8-OH-DPAT in goats, and 0.1 mg kg-1 R-8-OH-DPAT hydrobromide was administered intramuscularly (i.m.) and intravenously (i.v.) to six goats in a two-phase cross-over design experiment. Venous blood samples were collected from the jugular vein 2, 5, 10, 15, 20, 30, 40 and 60 min following treatment and analysed by liquid chromatography tandem mass spectrometry. Bioavailability and pharmacokinetic parameters were determined by a one-compartment analysis. Mean bioavailability of R-8-OH-DPAT when injected i.m. was 66%. The mean volume of distribution in the central compartment was 1.47 L kg-1 . The mean plasma body clearance was 0.056 L kg-1  min-1 . All goats injected i.v. and two of six goats injected i.m. showed signs of serotonin toxicity. In conclusion, R-8-OH-DPAT is well absorbed following i.m. injection and the observed pharmacokinetics suggest that administration via dart is feasible. Administration of R-8-OH-DPAT hydrobromide, at a dosage of 0.1 mg kg-1 , resulted in the observation of clinical signs of serotonin toxicity in the goats. It is suggested that dosages for the clinical use of the compound should be lower in order to achieve the desired clinical effect without causing serotonin toxicity.

The pharmacokinetics and pharmacological effect of (S)-5-OH-DPAT following controlled delivery with transdermal iontophoresis.

The pharmacokinetic (PK) and pharmacodynamic (PD) properties of the active (S)-enantiomer of the potent dopamine (DA) agonist 5-hydroxy-2-(N,N,-di-n-propylamino)tetralin (5-OH-DPAT) were investigated in a novel anesthetized animal model. First, the relationship between current density, in vivo transport, and plasma profile was characterized. Second, the effect of the anesthetic mixture, transdermal iontophoresis, and blood sampling on the striatal DA release (PD end point) was investigated. Third, the PK-PD relationship following transdermal iontophoresis was investigated during a controlled reversible pharmacological response. Given that striatal DA levels are unaltered during experimental procedures, this rat model can be used to investigate the PK-PD relationship. The in vivo flux was linearly correlated with the current density, indicating that drug delivery can be titrated by the current density. Following transdermal iontophoresis and intravenous infusion, a strong reversible effect was observed. Compartmental modeling showed that the relationship between plasma concentration and biomarker response is best characterized by an effect compartment, rather than an indirect response model. In addition, covariate analysis suggested that the delivery rate can affect the PD efficiency. Finally, PK-PD analysis revealed that steady delivery rates are translated into continuous dopaminergic stimulation. This can be of benefit for reducing side effects in the symptomatic treatment of Parkinson's disease with 5-OH-DPAT.

Pharmacokinetics and pharmacodynamics analysis of transdermal iontophoresis of 5-OH-DPAT in rats: in vitro-in vivo correlation.

Pharmacokinetics and dopaminergic effect of dopamine agonist 5-OH-DPAT in vivo were determined following transdermal iontophoresis in rats based on drug concentration in plasma (C(p)) and dopamine levels in striatum (C(DA)). Correlation of the in vitro transport with the pharmacokinetic-pharmacodynamic (PK-PD) profiles was characterized in the transport in dermatomed rat skin (DRS) and rat stratum corneum (RSC). The integrated in vivo PK-PD and in vitro transport models successfully described time course of C(p), C(DA), and in vitro flux in DRS and RSC. Population value of steady-state flux (J(ss)) in vivo (31 nmol/cm(2) . h with 95% confidence interval (CI) = 20-41) is closer to J(ss) in vitro in DRS (61 nmol/cm(2) . h, CI = 54-67) than in vitro J(ss) in RSC (98 nmol/cm(2) . h, CI = 79-117). On the other hand, skin release rate constant (K(R)) in vivo was similar to the K(R) in RSC (4.8/h, CI = 2.4-7.1 vs. 2.6/h, CI = 2.5-2.6). Kinetic lag time (t(L)) in vivo was negligible, which is close to in vitro t(L) in RSC (0.0 h, CI = 0.0-0.1). Based on nonlinear mixed-effect modeling, profiles of C(p) and C(DA) were successfully predicted using in vitro values of J(ss) in DRS with K(R) and t(L) in RSC. A considerable dopaminergic effect was achieved, indicating the feasibility to reach therapeutically effective concentrations of 5-OH-DPAT upon transdermal iontophoresis.

8-[3H]-hydroxy-2-(di-n-propylamino)tetralin binding sites in blood lymphocytes of rats and the modulation by mitogens and immobilization.

Serotonin 5-HT(1A) receptors were characterized in rat resting lymphocytes obtained by cardiac puncture with the use of the ligand [3H]8-hydroxy-2-(di-n-propylamino)tetralin. Selectivity of the specific binding was demonstrated by inhibition experiments with various serotonergic and nonserotonergic drugs. The rank order of potency for inhibition was WAY-100478>pindobind>NAN-190>buspirone>imipramine>serotonin. While pimozide, desipramine, nomifensine, haloperidol and sulpiride did not inhibit the binding. Kinetic parameters calculated from saturation experiments indicated one site of interaction, with an equilibrium dissociation constant of 2.50 nM and maximum binding capacity of 487.21 nmol/10(6) cells. Complete dissociation was obtained with serotonin as the displacement agent, and equilibrium dissociation constant calculated by association and dissociation experiments was 2.03 nM. Thus, serotonin 5-HT(1A) receptors are present in resting lymphocytes. The in vivo administration of the mitogens lipopolysacharide (0.1 mg/kg, 18 h) or concanavalin A (0.2 mg/kg, 18 h) increased the number of sites. The elevation produced by the latter was of higher magnitude than that of lipopolysacharide, and two sites of the binding were determined by isotopic dilution. Immobilization stress (1 h daily for 7 days) also resulted in a significant increase of binding capacity, but was smaller than that produced by the mitogens. The affinity of binding was not affect by the treatments. The results indicate that serotonin 5-HT(1A) receptors are modulated by unspecific and specific immune system activation, as well as by a potent stress condition, which might result in relevant functional modifications in the response of rat lymphocytes.

An integrative pharmacokinetic and pharmacodynamic study of the 5-HT1A receptor antagonist (S)-UH-301 in the rat.

(S)-UH-301 ((-)-(S)-5-fluoro-8-hydroxy-2-(di-n-propylamino)tetralin hydrochloride) is a well known 5-HT(1A) receptor antagonist. The present study describes the pharmacokinetic properties of (S)-UH-301 after subcutaneous administration in rats, using a newly developed HPLC-UV bioanalytical method. The relationships between (S)-UH-301 concentrations and some pharmacodynamic effects were also studied. The AUC of (S)-UH-301 in brain, but not in plasma, increased in proportion to dose (1-100 mumol/kg). However, at doses above 32 mumol/kg, peak concentrations of the drug did not increase in proportion to dose, and there was a doubling of its apparent half-life. There was a good correspondence between the time courses for the antagonism of 8-OH-DPAT-induced motor behaviours and hypothermia and the tissue concentrations of (S)-UH-301. Doses of (S)-UH-301 above 10 mumol/kg decreased 5-HT and dopamine synthesis. Therefore, a selective 5-HT(1A) antagonistic dose range of (S)-UH-301 should be 0.1-10 mumol/kg s.c., corresponding to concentrations below approximately 10 nmol/g in brain and approximately 1 nmol/ml in plasma.

A set-point model with oscillatory behavior predicts the time course of 8-OH-DPAT-induced hypothermia.

Agonists for the 5-hydroxytryptamine (HT)(1A) receptor induce a hypothermic response that is believed to occur by lowering of the body's set-point temperature. We have developed a physiological model that can be used to predict the complex time course of the hypothermic response after administration of 5-HT(1A) agonists to rats. In the model, 5-HT(1A) agonists exert their effect by changing heat loss through a control mechanism with a thermostat signal that is proportional to the difference between measured and set-point temperature. Agonists exert their effect in a direct concentration-dependent manner, with saturation occurring at higher concentrations. On the basis of simulations, it is shown that, depending on the concentration and the intrinsic efficacy of a 5-HT(1A) agonist, the model shows oscillatory behavior. The model was successfully applied to characterize the complex hypothermic response profiles after administration of the reference 5-HT(1A) agonists R-8-hydroxy-2-(di-n-propylamino)tetralin (R-8-OH-DPAT) and S-8-OH-DPAT. This analysis revealed that the observed difference in effect vs. time profile for these two reference agonists could be explained by a difference in in vivo intrinsic efficacy.

Rapid desensitization of 5-HT(1A) receptors in Fawn-Hooded rats after chronic fluoxetine treatment.

Anxiety, platelet serotonin (5-HT) content and functions of the 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) were measured in Sprague--Dawley (SD) and Fawn-Hooded (FH) rats, a strain with genetically impaired 5-HT storage and reuptake system and a putative model of depression and anxiety. In addition, the effects of 7 and 16 days treatment with the selective serotonin reuptake inhibitor (SSRI) fluoxetine on 8-OH-DPAT-induced responses were studied. FH rats showed significantly higher anxiety in the social interaction test, and much lower platelet 5-HT content compared to SD rats. The efficacy of 8-OH-DPAT (15-120 microg/kg, i.v.) to induce lower lip retraction (an effect mediated by median raphe receptors) was increased in FH rats. In most FH but only a few SD rats a special neurological syndrome, clonic movement of the masseters and in-and-out movement of the eyeballs, was induced by 8-OH-DPAT, and this behaviour like other effects of 8-OH-DPAT, was completely blocked by pretreatment with the 5-HT(1A) receptor antagonist WAY-100635. In SD rats fluoxetine (10 mg/kg/day, i.p.) caused a moderate inhibition of 8-OH-DPAT-induced hypothermia, an effect mediated most likely by hypothalamic 5-HT(1A) receptors, (-19% and -40% after 7 and 16 days of fluoxetine, 24 h after the last injection, respectively). In FH rats fluoxetine caused a rapid and complete reduction in the 8-OH-DPAT-induced hypothermia (-65% and -91% after 7 and 16 days of fluoxetine, respectively). Fluoxetine caused no change in lower lip retraction but a reduction in the masseter-eyeball syndrome in both SD and FH rats. Our data provide evidence that in FH rats, median raphe 5-HT(1A) receptors are hypersensitive, and the hypothalamic 5-HT(1A) receptor desensitization, caused by SSRI antidepressants, is faster and more complete. These data support the notion that chronic treatment with SSRIs induces a desensitization of some 5-HT(1A) receptor populations, and impaired 5-HT storage and reuptake may accelerate this process.

Enantioselective high-performance liquid chromatographic analysis of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin. Application to a pharmacokinetic-pharmacodynamic study in rats.

A rapid, sensitive and enantioselective HPLC assay for the simultaneous determination of the reference 5-HT1A receptor agonists, R-(+)- and S-(-)-8-hydroxy-2-(di-n-propylamino)tetralin (R-8-OH-DPAT and S-8-OH-DPAT, respectively), in rat blood is presented. A selective extraction procedure was developed using a preliminary sample clean-up followed by isolation of R- or S-8-OH-DPAT on mixed-mode NARC-2 solid-phase columns. Separation of the enantiomers was performed by high-performance liquid chromatography using a Chiracel OD-R column. Detection was obtained using an electrochemical detector set at a voltage of 0.63 V. The mobile phase consisted of a 50 mM phosphate buffer (pH 5.5)-acetonitrile (80:20, v/v) mixture. At a flow-rate of 1 ml min(-1), the total run time was approximately 14 min. The limit of detection for R- and S-8-OH-DPAT was 0.5 ng ml(-1). In the concentration range between 50 ng ml(-1) and 1000 ng ml(-1) intra- and inter-day relative standard deviations were less than 12%. The assay was applied to a pharmacokinetic-pharmacodynamic study in rats in which decrease of body temperature was used as a measure of 5-HT1A receptor-mediated effect. Values for clearance, volume of distribution at steady state and terminal elimination rate constant were 22+/-2 ml min(-1), 1969+/-473 ml and 156+/-34 min for R-8-OH-DPAT and 16+/-1 ml min(-1), 3353+/-347 ml and 334+/-36 min for S-8-OH-DPAT, respectively. No enantiomeric interconversion was observed in vivo from R-8-OH-DPAT to S-8-OH-DPAT or vice versa.

Nasal absorption of (S)-UH-301 and its transport into the cerebrospinal fluid of rats.

Targeting the brain via nasal administration of drugs has been studied frequently over the last few years. In this study, the serotonin-1a receptor antagonist (S)-5-fluoro-8-hydroxy-2-(dipropyl-amino) tetralin ((S)-UH-301) hydrochloride was used as a model substance. The systemic absorption and transport of (S)-UH-301 into male Sprague-Dawley rat cerebrospinal fluid (CSF) were investigated after nasal and intravenous administration. Blood and CSF samples were obtained at regular time intervals from the arteria carotis and by cisternal puncture, respectively, after administration to both nostrils (total 12 micromol/kg) or into the vena jugularis (6 micromol/kg). The concentrations of (S)-UH-301 in plasma and CSF were measured by HPLC with electrochemical detection. The maximum plasma concentration of intranasal (S)-UH-301 occurred in about 7 min and the absolute bioavailability seemed to be complete (F=1.2+/-0.4). Initially, no increased concentrations of (S)-UH-301 were seen in CSF after nasal compared to intravenous administration i.e. it appeared that no direct transport of (S)-UH-301 from the nasal cavity, along the olfactory neurons and into the CSF occurred. However, a prolonged duration of the concentration was seen after nasal administration of (S)-UH-301 and after about 20 min the CSF(na):CSF(iv) concentration ratio (corrected for different dosage) exceeded 1.

Determination of U-89968E, a 5HT1a agonist in rat plasma using solid-phase extraction, precolumn derivatization and reversed-phase high-performance liquid chromatography.

A selective and sensitive HPLC method was developed for the determination of U-39968E in rat plasma. The assay involved solid-phase extraction of the analyte and the internal standard and precolumn derivatization with cyclohexane-1,3-dione reagent before injection on to the HPLC column. The samples were chromatographed on a Spherisorb S5 CN column (25 cm x 4.6 mm i.d.) with a mobile phase containing acetonitrile-trifluoroacetic acid-water (17:0.2:83, v/v/v) at a flow rate of 1.5 ml min-1. The column eluent was monitored by flourescence detection with excitation at 272 nm and emission at 320 nm. The assay is linear over the range 4-759 ng ml-1. The relative standard deviation at the limit of quantification, 4 ng ml-1, was 7.1%. This method was successfully applied to the determination of U-89968E in rat plasma during pharmacokinetic studies.

Development of chiral high performance liquid chromatographic assays for the enantiomers of 8-hydroxy-(di-n-propylamino)tetralin and three 8-keto-pyrrole-substituted analogues.

Two chiral HPLC methods using protein-based stationary phases for the analysis of 8-hydroxy-(di-n-propylamino)tetralin and three 8-keto-pyrrole analogues are presented. Efficient solid-phase extraction enabled quantification of 0.02 microgram ml-1 of the 8-keto-pyrrole analogues from 500 microliters rat hepatocyte suspensions, and 0.2 microgram ml-1 to be measured from 50 microliters of rat plasma using UV detection at 315 nm.

Bioanalytical strategies to support a discovery research programme.

To facilitate the selection of drug candidates from a discovery research programme, a strategy was developed to assess the preliminary metabolism and pharmacokinetics of numerous chemical entities. A three-level "screening funnel" was set up, using both in-vitro and in-vivo techniques, requiring bioanalytical methods for the determination of parent compound. Simple high performance liquid chromatography (HPLC)/UV assays with minimal sample workup were adequate for the initial high throughput in-vitro screen used to assess in-vitro metabolic stability but a more selective sample extraction method was required for the second level of the screen. Here, rats were infused with drug to steady-state concentrations and whole blood, plasma, and brain tissue homogenate were analysed to assess clearance and to investigate blood/brain barrier penetration. Generally, HPLC/UV was adequate as only moderate sensitivities were required. However, the final level of the screen, a rat PO/IV bioavailability study, needed far more sensitive assays and often presented significant analytical challenges.