The effects of GR127935, a putative 5-HT1D receptor antagonist, on brain 5-HT metabolism, extracellular 5-HT concentration and behaviour in the guinea pig.

Studies of neurotransmitter release in guinea pig and human brain indicate that the 5-HT terminal autoreceptor is the 5-HT1D subtype and that it regulates the depolarization evoked release of 5-HT. Thus, blockade of the terminal 5-HT autoreceptor should enhance 5-HT release in vivo. In the present study, we have used the recently described, selective and potent 5-HT1D receptor antagonist, GR127935, to determine if blockade of the terminal 5-HT autoreceptor enhanced 5-HT neurotransmission in the guinea pig. Neurochemical studies showed that GR127935 (0.1, 0.3 and 1.0 mg/kg i.p.) significantly increased 5-HT metabolism in forebrain regions but not in the raphe nucleus of the guinea pig. However, using in vivo dialysis, GR127935 did not significantly increase cortical 5-HT efflux when given either systemically (1 and 5 mg/kg i.p.) or by infusion via the probe directly into the cortex (10, 33 and 100 microM). Fast cyclic voltammetry studies in the guinea pig dorsal raphe slice in vitro failed to observe any significant effects of GR127935 (0.01-1 microM) on electrically evoked 5-HT release. Behavioural studies in the guinea pig were also unable to demonstrate any effects of GR127935 (0.1-3.0 mg/kg i.p.) per se or in combination with the 5-HT precursor 5-hydroxytryptophan. Taken together, results from the present neurochemical and behavioral studies in the guinea pig provide little substantial evidence that blockade of the terminal 5-HT autoreceptor following the acute administration of GR127935 increased brain 5-HT neurotransmission in vivo.

The behavioural and neurochemical profile of the putative dopamine D3 receptor agonist, (+)-PD 128907, in the rat.

The functional relevance of the dopamine D3 receptor is still unresolved, largely because of the absence of selective D3 receptor ligands. In the present study we have examined the in vivo profile of (+)-PD 128907, a potent and functionally selective D3 receptor agonist. Low doses of (+)-PD 128907 reduced spontaneous locomotor activity in the rat (ED50 = 13 +/- 3 micrograms/kg, s.c.) a response which was comparable with the non-selective D2,3 receptor agonist apomorphine (ED50 = 13 +/- 1.6 micrograms/kg, s.c.). In addition (+)-PD 128907 impaired prepulse inhibition of the acoustic startle response, with significant effects observed at doses of 30 micrograms/kg when appropriate prepulse intensities were used. Higher doses reversed gamma-butyrolactone-induced catecholamine synthesis (ED50 = 95 +/- 22 and 207 +/- 37 micrograms/kg in accumbens and striatum respectively) and induced yawning (100-300 micrograms/kg), penile grooming (30-1000 micrograms/kg) and sniffing (> or = 300 micrograms/kg) although doses 3- to 10-fold greater than apomorphine were required to produce maximal effects. In contrast to apomorphine, however, (+)-PD 128907 failed to induce intense stereotyped licking and biting in the rat. In view of the potency and selectivity of (+)-PD 128907 for the D3 receptor, a role in the control of locomotor activity is suggested. In addition, the observation that (+)-PD 128907 disrupts prepulse inhibition, a phenomenon which is also impaired in schizophrenic subjects, may indicate the pathological importance of this receptor subtype.

Identification of 3,5-dihydro-2-aryl-1H-pyrazolo[3,4-c]quinoline-1,4(2H)-diones as novel high-affinity glycine site N-methyl-D-aspartate antagonists.

Almost all of the existing known antagonists at the glycine site of the N-methyl-D-aspartate (NMDA) receptor have a low propensity for crossing the blood-brain barrier. It has been suggested that in many cases this may be due to the presence of a carboxylic acid which is a common feature of most of the potent full antagonists at this receptor. In this study, 2-aryl-1H-pyrazolo[3,4-c]quinoline-1,4(2H)-diones were found to have high-affinity binding at the glycine receptor. In particular, structure-activity studies identified 7-chloro-3,5-dihydro-2-(4-methoxyphenyl)-1H-pyrazolo[3,4-c]quinoline- 1,4(2H)-dione as the most potent of a series of analogues with an IC50 of 3.3 nM. The measured pKa values in this class of compounds (typically 4.0) indicate they are of equivalent acidity to carboxylic acids. Functional antagonism was demonstrated by inhibition of NMDA-evoked responses in rat cortical slices. Anticonvulsant activity in DBA/2 mice was achieved after dosing by direct injection into the cerebral ventricles, but no activity was seen after systemic administration, suggesting low brain penetration with this class of antagonists.

Effect of plasma protein binding on in vivo activity and brain penetration of glycine/NMDA receptor antagonists.

A major issue in designing drugs as antagonists at the glycine site of the NMDA receptor has been to achieve good in vivo activity. A series of 4-hydroxyquinolone glycine antagonists was found to be active in the DBA/2 mouse anticonvulsant assay, but improvements in in vitro affinity were not mirrored by corresponding increases in anticonvulsant activity. Here we show that binding of the compounds to plasma protein limits their brain penetration. Relative binding to the major plasma protein, albumin, was measured in two different ways: by a radioligand binding experiment or using an HPLC assay, for a wide structural range of glycine/NMDA site ligands. These measures of plasma protein binding correlate well (r = 0.84), and the HPLC assay has been used extensively to quantify plasma protein binding. For the 4-hydroxyquinolone series, binding to plasma protein correlates (r = 0.92) with log P (octanol/pH 7.4 buffer) over a range of log P values from 0 to 5. The anticonvulsant activity increases with in vitro affinity, but the slope of a plot of pED50 versus pIC50 is low (0.40); taking plasma protein binding into account in this plot increases the slope to 0.60. This shows that binding to albumin in plasma reduces the amount of compound free to diffuse across the blood-brain barrier. Further evidence comes from three other experiments: (a) Direct measurements of brain/blood ratios for three compounds (2, 16, 26) show the ratio decreases with increasing log R. (b) Warfarin, which competes for albumin binding sites dose-dependently, decreased the ED50 of 26 for protection against seizures induced by NMDLA. (c) Direct measurements of brain penetration using an in situ brain perfusion model in rat to measure the amount of drug crossing the blood-brain barrier showed that compounds 2, 26, and 32 penetrate the brain well in the absence of plasma protein, but this is greatly reduced when the drug is delivered in plasma. In the 4-hydroxyquinolones glycine site binding affinity increases with lipophilicity of the 3-substituent up to a maximum at a log P around 3, then does not improve further. When combined with increasing protein binding, this gives a parabolic relationship between predicted in vivo activity and log P, with a maximum log P value of 2.39. Finally, the plasma protein binding studies have been extended to other series of glycine site antagonists, and its is shown that for a given log P these have similar protein binding to the 4-hydroxyquinolones, except for compounds that are not acidic. The results have implications for the design of novel glycine site antagonists, and it is suggested that it is necessary to either keep log P low or pKa high to obtain good central nervous system activity.

4-substituted-3-phenylquinolin-2(1H)-ones: acidic and nonacidic glycine site N-methyl-D-aspartate antagonists with in vivo activity.

4-Substituted-3-phenylquinolin-2(1H)-ones have been synthesized and evaluated in vitro for antagonist activity at the glycine site on the NMDA (N-methyl-D-aspartate) receptor and in vivo for anticonvulsant activity in the DBA/2 strain of mouse in an audiogenic seizure model. 4-Amino-3-phenylquinolin-2(1H)-one (3) is 40-fold lower in binding affinity but only 4-fold weaker as an anticonvulsant than the acidic 4-hydroxy compound 1. Methylsulfonylation at the 4-position of 3 gives an acidic compound (6, pKa = 6.0) where affinity is fully restored but in vivo potency is significantly reduced (Table 1). Methylation at the 4-position of 1 to give 18 results in the abolition of measurable affinity, but the attachment of neutral hydrogen bond-accepting groups to the methyl group of 18 produces compounds with comparable in vitro and in vivo activity to 1 (e.g., 23 and 28, Table 2). Replacement of the 4-hydroxy group of 1 with an ethyl group abolishes activity (42), but again, incorporation of neutral hydrogen bond acceptors to the terminal carbon atom restores affinity (e.g., 36, 39, and 40, Table 3). Replacement of the 4-hydroxy group of the high-affinity compound 2 with an amino group produces a compound with 200-fold reduced affinity (43; IC50 = 0.42 microM, Table 4) which is nevertheless still 10-fold higher in affinity than 3. The results in this paper indicate that anionic functionality is not an absolute requirement for good affinity at the glycine/NMDA site and provide compelling evidence for the existence of a ligand/receptor hydrogen bond interaction between an acceptor attached to the 4-position of the ligand and a hydrogen bond donor attached to the receptor.

Pharmacokinetics of valganciclovir and ganciclovir following multiple oral dosages of valganciclovir in HIV- and CMV-seropositive volunteers.

OBJECTIVE: Ganciclovir is commonly used in the treatment of cytomegalovirus (CMV) disease in patients who are immunocompromised and for the prevention of CMV disease in solid organ transplant recipients. Owing to limited bioavailability and saturable absorption, the use of oral ganciclovir in CMV retinitis is restricted to maintenance therapy only. As induction therapy must be given intravenously, an oral formulation which could be used for induction would offer significant benefits. A previous study of valganciclovir, a valyl ester prodrug of ganciclovir showed a 10-fold increase in plasma ganciclovir concentrations compared with the oral formulation. However, before studies can be conducted to confirm the utility of oral valganciclovir for the treatment and prevention of CMV disease, a dose must be selected for use in these studies. This study was designed to investigate the pharmacokinetics of ganciclovir and valganciclovir. DESIGN AND PARTICIPANTS: The study was an open-label, randomised, 4-way crossover, dose-ranging pharmacokinetic study, conducted in 39 patients who were HIV- and CMV-seropositive. The participants were randomised to one of 2 groups: fasted (n = 19) and fed (n = 20). In both groups, participants received 450, 875, 1750 and 2625 mg oral valganciclovir once daily for 3 days in a randomised order. RESULTS: In the 32 participants who completed the study, valganciclovir was rapidly absorbed and converted into ganciclovir (maximum ganciclovir concentrations occurred after 1.0 to 1.75 hours in the fasted group and 1.5 to 2.0 hours in the fed group). Systemic exposure to valganciclovir was low [with an area under the concentration-time curve to 24 hours (AUC24) of 1.3 to 2.5% that of ganciclovir]. The mean plasma concentrations of ganciclovir were dose-related. Peak concentrations of ganciclovir were achieved approximately 30 minutes after those for valganciclovir. In the fed state, the AUC24 of ganciclovir increased proportionally with dose. The mean AUC24 values for ganciclovir were slightly higher following food (24 to 56%) than in the fasted state. Based on linear regression of AUC24 values from the fed group, a dose of valganciclovir of 900 mg/day is expected to produce a daily exposure (AUC24) comparable with an intravenous dose of ganciclovir 5 mg/kg/day. CONCLUSIONS: These results show that once daily oral valganciclovir can produce exposures of ganciclovir (AUC24) exceeding those attained using intravenous ganciclovir 10 mg/kg. This suggests that oral valganciclovir may be suitable in many circumstances currently requiring intravenous ganciclovir, allowing for more convenience in the management of patients with CMV retinitis by utilising a 2 or 4 tablet daily regimen to cover all phases of treatment.

The anticonvulsant and behavioural profile of L-687,414, a partial agonist acting at the glycine modulatory site on the N-methyl-D-aspartate (NMDA) receptor complex.

1. The anticonvulsant and behavioural effects of the glycine/NMDA receptor partial agonist, L-687,414 (R(+)-cis-beta-methyl-3-amino-1-hydroxypyrrolid-2-one) have been investigated in rodents. 2. L-687,414 dose-dependently antagonized seizures induced by N-methyl-D,L- aspartic acid (NMDLA, ED50 = 19.7 mg kg-1), pentylenetetrazol (PTZ, ED50 = 13.0 mg kg-1) and electroshock (ED50 = 26.1 mg kg-1) when given intravenously 15 min before test, in male Swiss Webster mice but was most potent against audiogenic seizures induced by a 120 dB bell in DBA/2 mice (ED50 = 5.1 mg kg-1, i.p., 30 min before test). 3. L-687,414 also induced impairments of performance in a rotarod test in both Swiss Webster and DBA/2 mice and the ratio [rotarod MED:anticonvulsant ED50] varied between 0.9 and 5, depending on the convulsant used. 4. Similar behaviours to those seen after administration of the non-competitive NMDA receptor antagonist, MK-801 (head weaving, body rolling, hyperlocomotion) were seen in the mouse after giving L-687,414, although the peak effect occurred at a dose (100 mg kg-1) which was 5-20 times the anticonvulsant ED50S, depending on the convulsant used. Unlike MK-801, however, doses of L-687,414 that were behaviourally stimulant did not increase dopamine turnover in the nucleus accumbens. 5. Consistent with the interaction of L-687,414 with the glycine/NMDA receptor, the anticonvulsant, ataxic and motor stimulant effects of the compound were significantly attenuated by the glycine/NMDA receptor agonist, D-serine (10-100 micrograms per mouse, i.c.v.). 6. The results show that L-687,414 is a potent, orally active anticonvulsant with a more benign pharmacological profile than antagonists acting at the ion channel of the NMDA receptor complex. The compound is a useful tool with which to probe the functional role of the glycine co-agonist site in vivo.

The glycine/NMDA receptor antagonist, L-701,324 reverses isolation-induced deficits in prepulse inhibition in the rat.

Previous studies have demonstrated that the glycine/NMDA receptor antagonist, L-701,324 (7-chloro-4-hydroxy-3(3-phenoxy)phenyl-2(H)quinolone) blocks the activation of mesolimbic dopamine systems induced following psychostimulant administration in the rat (Bristow et al. 1994). In the present study, pretreatment with L-701,324 also reversed the deficit in prepulse inhibition (PPI) observed in rats reared in social isolation after weaning. Given that PPI is also attenuated in schizophrenic patients and that isolation rearing induces both neurochemical and behavioural abnormalities suggestive of a physiologically induced state of dopaminergic hyperactivity, these results suggest that blockade of the glycine/NMDA receptor may offer a new strategy for the development of novel antipsychotic agents.

Rat strain differences in the ability to disrupt sensorimotor gating are limited to the dopaminergic system, specific to prepulse inhibition, and unrelated to changes in startle amplitude or nucleus accumbens dopamine receptor sensitivity.

Previous studies indicate that a variety of pharmacological agents interfere with the prepulse inhibition of the acoustic startle (PPI) response including phencyclidine (PCP), 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), amphetamine, and apomorphine. Strain differences have been observed in the ability of apomorphine to disrupt PPI, although the degree to which these strain differences occur after administration of nondopaminergic drugs or the degree to which differences can be observed in other models of dopamine (DA) receptor activation has not been elucidated. The present study tested the effects of apomorphine, amphetamine, 8-OH-DPAT, and PCP on PPI in the Sprague Dawley and Wistar rat strains. Because apomorphine disrupts PPI via activation of DA receptors in the nucleus accumbens, apomorphine-induced hyperlocomotion, also a behavioral model of nucleus accumbens DA receptor activation, was measured in both rat strains. Administration of PCP or 8-OH-DPAT attenuated PPI in both strains, whereas apomorphine and amphetamine only attenuated PPI in Wistar rats. The ability of apomorphine to increase motor activity in the absence of a startle-eliciting stimulus was similar in the two strains, as was apomorphine-induced hyperlocomotion. A time course analysis of the effects of apomorphine on startle response in Sprague Dawley rats found that changes in the magnitude of PPI followed changes in basic startle amplitude. Similarly, no apomorphine-induced attenuation of PPI was observed in Sprague Dawley rats after 6-OHDA-induced DA receptor supersensitivity in the nucleus accumbens. These data suggest a dissociation between the effects of DA receptor agonists in PPI and other behavioral models of DA receptor activation.

L-745,870, a subtype selective dopamine D4 receptor antagonist, does not exhibit a neuroleptic-like profile in rodent behavioral tests.

This study examined the high-affinity, selective dopamine D4 receptor antagonist, L-745,870 (3-([4-(4-chlorophenyl)piperazin-1-yl]methyl)-1H-pyrrolo[2, 3-b]pyridine) in rodent behavioral models used to predict antipsychotic potential and side-effect liabilities in humans. In contrast to the classical neuroleptic, haloperidol, and the atypical neuroleptic, clozapine, L-745,870 failed to antagonize amphetamine-induced hyperactivity in mice or impair conditioned avoidance responding in the rat at doses selectively blocking D4 receptors. Furthermore, L-745,870 failed to reverse the deficit in prepulse inhibition of acoustic startle responding induced by the nonselective dopamine D2/3/4 receptor agonist apomorphine, an effect which was abolished in rats pretreated with the D2/3 receptor antagonist, raclopride (0.2 mg/kg s.c.). L-745,870 had no effect on apomorphine-induced stereotypy in the rat but did induce catalepsy in the mouse, albeit at a high dose of 100 mg/kg, which is likely to occupy dopamine D2 receptors in vivo. High doses also impaired motor performance; in rats L-745,870 significantly reduced spontaneous locomotor activity (minimum effective dose = 30 mg/kg) and in mice, L-745,870 reduced the time spent on a rotarod revolving at 15 rpm (minimum effective dose = 100 mg/kg). Altogether these results suggest that dopamine D4 receptor antagonism is not responsible for the ability of clozapine to attenuate amphetamine-induced hyperactivity and conditioned avoidance responding in rodents. Furthermore, the lack of effect of L-745,870 in these behavioral tests is consistent with the inability of the compound to alleviate psychotic symptoms in humans.

Anticonvulsant and behavioral profile of L-701,324, a potent, orally active antagonist at the glycine modulatory site on the N-methyl-D-aspartate receptor complex.

The anticonvulsant and behavioral profile of the glycine/N-methyl-D-aspartate receptor antagonist L-701,324 [7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(H)quinolone] has been examined in rodents. In mice, L-701,324 protected against seizures induced by N-methyl-DL-aspartate (ED50 = 3,4 mg/kg i.v.), pentylenetetrazol (ED50 = 2.8 mg/kg i.v.) and electroshock (ED50 = 1.4 mg/kg i.v.) but was most potent against audiogenic seizures in DBA/2 mice (ED50 = 0.96 mg/kg i.p.). L-701,324 was also active p.o. in mice (ED50 = 1.9,6.7, 20.7 and 34 mg/kg against audiogenic, electroshock-induced, N-methyl-DL-aspartate-induced and pentylenetetrazol-induced seizures, respectively) but showed weaker anticonvulsant activity in rats (ED50 = 90.5 mg/kg p.o., compared with 2.3 mg/kg i.v., against pentylenetetrazol-induced seizures), most probably because of the lower brain concentrations achieved in this species. Although anticonvulsant activity was also associated with impaired rotarod performance, L-701,324 failed to significantly increase locomotor activity or dopamine turnover in the nucleus accumbens at doses of up to 10 mg/kg i.v. in mice. Thus, in contrast to N-methyl-D-aspartate receptor ion channel blockers such as MK-801 (dizocilpine), L-701,324 is a potent, p.o. active anticonvulsant with a reduced propensity to activate mesolimbic dopaminergic systems in rodents.