The PET tracer [11C]MK-6884 quantifies M4 muscarinic receptor in rhesus monkeys and patients with Alzheimer's disease.

Positron emission tomography (PET) ligands play an important role in the development of therapeutics by serving as target engagement or pharmacodynamic biomarkers. Here, we describe the discovery and translation of the PET tracer [11C]MK-6884 from rhesus monkeys to patients with Alzheimer's disease (AD). [3H]MK-6884/[11C]MK-6884 binds with high binding affinity and good selectivity to an allosteric site on M4 muscarinic cholinergic receptors (M4Rs) in vitro and shows a regional distribution in the brain consistent with M4R localization in vivo. The tracer demonstrates target engagement of positive allosteric modulators of the M4R (M4 PAMs) through competitive binding interactions. [11C]MK-6884 binding is enhanced in vitro by the orthosteric M4R agonist carbachol and indirectly in vivo by the acetylcholinesterase inhibitor donepezil in rhesus monkeys and healthy volunteers, consistent with its pharmacology as a highly cooperative M4 PAM. PET imaging of [11C]MK-6884 in patients with AD identified substantial regional differences quantified as nondisplaceable binding potential (BPND) of [11C]MK-6884. These results suggest that [11C]MK-6884 is a useful target engagement biomarker for M4 PAMs but may also act as a sensitive probe of neuropathological changes in the brains of patients with AD.

Population pharmacokinetics of pegaptanib sodium (Macugen(®)) in patients with diabetic macular edema.

OBJECTIVE: Population pharmacokinetic modeling of pegaptanib was undertaken to determine influence of renal function on apparent clearance. METHODS: In a randomized, double-masked multicenter trial, intravitreal pegaptanib (0.3, 1.0, or 3.0 mg/eye) was administered in patients with diabetic macular edema every 6 weeks for 12-30 weeks. A one-compartment model with first-order absorption, distribution volume, and clearance was used to characterize the pegaptanib plasma concentration-time profile. RESULTS: In 58 patients, increases in area under the concentration-time curve (AUC) to end of the dosing interval (AUC0-tau) and maximum concentration with repeat doses were <6%, indicating minimal plasma accumulation. Sex and race did not have clinically significant effects on pegaptanib exposure. In the final model, the AUC extrapolated to infinite time and maximum concentration increased by ≥50% in older patients (aged >68 years) relative to younger patients due to decreases in creatinine clearance (CRCL), a significant predictor of clearance. Pegaptanib clearance was reduced by 29% when CRCL decreased by 50%. The change in exposure with CRCL (range, 0-190 mL/minute) was < 10-fold with 0.3-3.0 mg doses. CONCLUSION: While pegaptanib clearance and AUC were significantly influenced by CRCL, the predicted exposure in patients with renal insufficiency or renal failure shows no evidence that a dose adjustment is warranted, given the tenfold margin of safety observed over the dose range of 0.3-3.0 mg.

Integrating disease progression models, non-clinical pharmacokinetic data and treatment response endpoints to optimize intravitreal dosing regimens.

OBJECTIVE: To rapidly identify patients who will ultimately respond to 1 year of therapy, and optimize their inter dose interval. MATERIALS AND METHODS: An intravitreal (IVT) ophthalmic dosing paradigm was designed based on clinical efficacy, nonclinical pharmacokinetics (PK), and disease progression modeling. Relevant non-clinical PK models were used to extrapolate IVT drug concentrations to patients. RESULTS: Modeling predicted that > 80% of patients who would respond to 1 year of IVT treatment with an improvement in best-corrected visual acuity (BCVA) could be identified after the first 2 doses of treatment. These 2 initial doses produced ~ 75% of the maximum improvement in BCVA attainable. Moreover, the models also predicted those patients who responded after 1 year of treatment may tolerate an extension of the inter dose interval to 12 weeks without significant deterioration of BCVA. In contrast, > 70% of responsive patients who did not respond to 1 year of treatment showed inadequate responses after 2 doses. CONCLUSIONS: These models use data from 2 doses to identify those patients likely to benefit after 1 year of treatment, and thereafter can lengthen their inter dose interval without deleterious effects. This method identifies potential treatment responders early, and lengthens the inter dose interval during long-term administration while allowing non-responders to pursue alternative therapies earlier, thereby minimizing risk to the patient.

RTP801 gene expression is differentially upregulated in retinopathy and is silenced by PF-04523655, a 19-Mer siRNA directed against RTP801.

PURPOSE: The intraocular pharmacodynamics of PF-04523655, a small-interfering RNA (siRNA) directed against RTP801, was characterized using rat models of retinopathy. METHODS: Rat models of streptozotocin-induced diabetes and wet AMD were used to determine the onset, extent, and duration of siRNA inhibition of retinal RTP801 expression by PF-04523655, and this inhibition was characterized by pharmacokinetic/pharmacodynamic (PK/PD) modeling. A rat model of wet AMD was also used to examine PF-04523655 dose-dependent effects on the incidence of clinical grade 3 or 4 choroidal neovascularization lesions. Whole homogenate versus laser-capture microdissected (LCM) retinal samples were analyzed by quantitative PCR for RTP801 expression. RESULTS: RTP801 expression in RPE/choroid (RPE/C) increased in diabetic rats by up to 70% above nondiabetic rat levels. Inhibition of retinal RTP801 expression by PF-04523655 began 1 day after intravitreous injection and was observed through day 7 in the neurosensory retina and through day 14 or longer in RPE/C. PF-04523655 inhibition of RTP801 expression was maintained well after clearance of PF-04523655 from the eye and was best characterized by an effect compartment PK/PD model. Moreover, PF-04523655 administration decreased the incidence of clinical grade 3 or 4 lesions by approximately 60% (P = 0.053), and dose-dependently inhibited retinal RTP801 expression (P < 0.01). RTP801 expression was enriched in the outer nuclear layer in LCM samples. CONCLUSIONS: In rodent retinopathy models, administration of the siRNA, PF-04523655, reduced RTP801 expression in the retina, consistent with the RNA-induced silencing complex (RISC) mechanism of action. The pharmacodynamic profile from the animal models could be useful to elucidate dose and exposure dependency of RTP801 expression inhibition by siRNA.

Gene expression profiling of transporters in the solute carrier and ATP-binding cassette superfamilies in human eye substructures.

The barrier epithelia of the cornea and retina control drug and nutrient access to various compartments of the human eye. While ocular transporters are likely to play a critical role in homeostasis and drug delivery, little is known about their expression, localization and function. In this study, the mRNA expression levels of 445 transporters, metabolic enzymes, transcription factors and nuclear receptors were profiled in five regions of the human eye: cornea, iris, ciliary body, choroid and retina. Through RNA expression profiling and immunohistochemistry, several transporters were identified as putative targets for drug transport in ocular tissues. Our analysis identified SLC22A7 (OAT2), a carrier for the antiviral drug acyclovir, in the corneal epithelium, in addition to ABCG2 (BCRP), an important xenobiotic efflux pump, in retinal nerve fibers and the retinal pigment epithelium. Collectively, our results provide an understanding of the transporters that serve to maintain ocular homeostasis and which may be potential targets for drug delivery to deep compartments of the eye.

Dose-ranging evaluation of intravitreal siRNA PF-04523655 for diabetic macular edema (the DEGAS study).

PURPOSE: To evaluate the safety and efficacy of three doses of PF-04523655, a 19-nucleotide methylated double stranded siRNA targeting the RTP801 gene, for the treatment of diabetic macular edema (DME) compared to focal/grid laser photocoagulation. METHODS: This multicenter, prospective, masked, randomized, active-controlled, phase 2 interventional clinical trial enrolled 184 DME patients with best corrected visual acuity (BCVA) of 20/40 to 20/320 inclusive in the study eye. Patients were randomly assigned to 0.4-mg, 1-mg, 3-mg PF-04523655 intravitreal injections or laser. The main outcome measure was the change in BCVA from baseline to month 12. RESULTS: All doses of PF-04523655 improved BCVA from baseline through month 12. At month 12, the PF-04523655 3-mg group showed a trend for greater improvement in BCVA from baseline than laser (respectively 5.77 vs. 2.39 letters; P = 0.08; 2-sided α = 0.10). The study was terminated early at month 12 based on predetermined futility criteria for efficacy and discontinuation rates. PF-04523655 was generally safe and well-tolerated, with few adverse events considered treatment-related. By month 12, the discontinuation rates in the PF-04523655 groups were higher than the laser group and were inversely related to dose levels. CONCLUSIONS: PF-04523655 showed a dose-related tendency for improvement in BCVA in DME patients. Studies of higher doses are planned to determine the optimal efficacious dose of PF-04523655. PF-04523655 may offer a new mode of therapeutic action in the management of DME. (ClinicalTrials.gov number, NCT00701181.).

Amitifadine, a triple monoamine uptake inhibitor, reduces binge drinking and negative affect in an animal model of co-occurring alcoholism and depression symptomatology.

The co-occurrence of alcoholism and depression is highly prevalent and difficult to treat. In an animal model of binge drinking that exhibits abstinence-induced behaviors reminiscent of negative affective states, the triple monoamine uptake inhibitor, amitifadine, produced a selective, dose dependent attenuation of binge drinking. Amitifadine also reversed abstinence-induced increases in the intracranial self-stimulation threshold, a model of anhedonia, and immobility in the forced swim test, reflecting behavioral despair. In view of the safety profile of amitifadine in humans, including low risk for weight gain, lack of sexual side effects, and low potential for abuse, we hypothesize that amitifadine will be effective in treating co-occurring alcoholism and depression.

Population pharmacokinetics of pegaptanib in patients with neovascular, age-related macular degeneration.

The anti-vascular endothelial growth factor (VEGF) aptamer pegaptanib is eliminated primarily by renal clearance. Because renal function declines with age, pegaptanib exposure in patients with age-related macular degeneration (AMD) may increase. Therefore, a population pharmacokinetic (PK) analysis of pegaptanib was undertaken in Western and Asian AMD patients to determine the influence of renal function on apparent pegaptanib clearance (CL). Pegaptanib (0.3-3 mg per eye) was administered every 4 to 6 weeks to 262 AMD patients in 4 studies. Pegaptanib exposures (area under the concentration-time curve [AUC] and maximum plasma concentration) after 8 doses were similar to exposures following the first dose, consistent with the absence of plasma accumulation. A 1-compartment model parameterized in terms of the absorption rate constant, apparent volume of distribution, and CL was used to describe the pegaptanib plasma concentration data. Creatinine clearance (CLCR), body weight (WT), and age influenced pegaptanib PK. Decreasing CLCR from 70 to 30 mL/min doubled AUC. After adjustment for CLCR, WT, and age, the model predicted no race differences in CL or AUC. Given that the therapeutic 0.3 mg per eye dose of pegaptanib results in exposures one-tenth of those observed following the well-tolerated 3-mg dose, these results suggest that no dose adjustment is warranted for AMD patients with moderate renal insufficiency (CLCR >30 mL/min).

Effects of the triple monoamine uptake inhibitor DOV 102,677 on alcohol-motivated responding and antidepressant activity in alcohol-preferring (P) rats.

BACKGROUND: Concurrent inhibitors of dopamine, norepinephrine, and serotonin uptake have been proposed as novel antidepressants. Given the high comorbidity between alcoholism and depression, we evaluated the activity of DOV 102,677 (DOV) on alcohol-maintained responding and performance in the forced swim test (FST), a model of antidepressant (AD) activity, using alcohol-preferring (P) rats. METHODS: Following training to lever press for either alcohol (10% v/v) or sucrose (3, 2%, w/v) on a fixed-ratio 4 (FR4) schedule, DOV (1.56 to 50 mg/kg; PO) was given 25 minutes or 24 hours prior to evaluation. The effects of DOV (12.5 to 50 mg/kg; PO) in the FST were evaluated 25 minutes posttreatment. RESULTS: DOV (6.25 to 50 mg/kg) dose-dependently reduced alcohol-maintained responding by 59 to 88% at 25 minutes posttreatment, without significantly altering sucrose responding. The reduction in alcohol responding (44% at 50 mg/kg) was sustained for up to 120 hours after a single dose. Administration of a single dose of DOV (25, 50 mg/kg) 24 hours before testing suppressed alcohol responding for 48 hours by 59 to 62%. DOV (12.5 to 50 mg/kg) also dose-dependently reduced immobility of P rats in the FST. CONCLUSIONS: DOV produces both prolonged and selective reductions of alcohol-motivated behaviors in P rats. The elimination kinetics of DOV suggests that its long duration of action may be due to an active metabolite. DOV also produced robust AD-like effects in P rats. We propose that DOV may be useful in treating comorbid alcoholism and depression in humans.

Intravitreal concentrations of a near-infrared fluorescence-labeled biotherapeutic determined in situ using confocal scanning laser ophthalmoscopy.

PURPOSE: The pharmacokinetics of ophthalmic biotherapeutics are difficult to determine in human vitreous humor. Because of the high transparency of living tissue to near-infrared (NIR) light, the temporal changes in vitreous concentrations of a biomolecule labeled with an NIR fluorescent probe can be monitored in situ with a scanning laser ophthalmoscope (SLO). METHODS: A humanized IgG was labeled with the NIR probe IRDye800CW (CVX-4164). Rabbits were given CVX-4164 intravitreally, and NIR fluorescence intensity was measured in the central plane of the vitreous humor with an SLO. Fluorescence intensities were converted to concentrations by using standard curves. RESULTS: Little background fluorescence was detected, and the minimum detectable concentration of CVX-4164 was <10 nM. Vitreal concentrations of CVX-4164 determined in situ declined with time, with C(max) ≈ 1 µM and t½ = 145 hours (112-µg dose). The t½ of CVX-4164 was approximately three times greater than that of the IRDye800CW alone, whereas the vitreal clearance (CL) and volume of distribution (V(ss)) of the native dye were approximately 2000- and 550-fold greater than that of the conjugate. CVX-4164 concentrations determined in situ were 2.6 to 4.4 times higher than those determined by ex vivo NIR fluorescence or ELISA in homogenized vitreous humor, reflecting the greater spatial resolution of in situ imaging. Moreover, vitreal concentrations determined in situ were >3 orders of magnitude greater than plasma concentrations of CVX-4164, as determined by ELISA, and had a different kinetic profile. CONCLUSIONS: This study demonstrates the feasibility of determining the pharmacokinetics of intraocular biotherapeutics labeled with NIR fluorescent probes by in situ monitoring.

Preclinical evaluation of the abuse potential of the analgesic bicifadine.

The abuse liability of the analgesic bicifadine was investigated in animal models used to predict the abuse potential of psychostimulants in humans. Bicifadine, cocaine, d-amphetamine, bupropion, and desipramine were evaluated for the production of cocaine-like discriminative stimulus effects in rats. Cocaine, d-amphetamine, and bupropion dose-dependently and fully substituted for cocaine. Bicifadine and desipramine produced a maximum mean cocaine-lever selection of 80 and 69%, respectively, but doses yielding peak substitution strongly suppressed response rates. Microdialysis studies in normal waking rats indicated that d-amphetamine increased dopamine levels in the nucleus accumbens and striatum to a much greater degree than bicifadine, but bicifadine increased 5-hydroxytryptamine levels in the nucleus accumbens and striatum more than d-amphetamine. Bicifadine was also tested for intravenous self-administration in rhesus monkeys experienced with cocaine administration. Reinforcing effects of bicifadine were observed in only two of four subjects, whereas cocaine, d-amphetamine, and bupropion served as reinforcers in all four monkeys. When evaluated under a progressive ratio procedure, no dose of bicifadine maintained responding to the extent of cocaine, d-amphetamine, or bupropion. The discriminative stimulus effects associated with bicifadine were similar, but not identical, to those of psychostimulants. Although bicifadine maintained self-administration behavior in some subjects, its reinforcing efficacy was very low relative to cocaine, d-amphetamine, and bupropion. These results are consistent with the microdialysis findings of lower dopamine levels and higher 5-hydroxytryptamine levels after administration of bicifadine relative to d-amphetamine. Overall, the current findings support a low abuse potential of bicifadine, more resembling that of antidepressants than psychostimulants.

Pharmacological Properties of DOV 315,090, an ocinaplon metabolite.

BACKGROUND: Compounds targeting the benzodiazepine binding site of the GABAA-R are widely prescribed for the treatment of anxiety disorders, epilepsy, and insomnia as well as for pre-anesthetic sedation and muscle relaxation. It has been hypothesized that these various pharmacological effects are mediated by different GABAA-R subtypes. If this hypothesis is correct, then it may be possible to develop compounds targeting particular GABAA-R subtypes as, for example, selective anxiolytics with a diminished side effect profile. The pyrazolo[1,5-a]-pyrimidine ocinaplon is anxioselective in both preclinical studies and in patients with generalized anxiety disorder, but does not exhibit the selectivity between alpha1/alpha2-containing receptors for an anxioselective that is predicted by studies using transgenic mice. RESULTS: We hypothesized that the pharmacological properties of ocinaplon in vivo might be influenced by an active biotransformation product with greater selectivity for the alpha2 subunit relative to alpha1. One hour after administration of ocinaplon, the plasma concentration of its primary biotransformation product, DOV 315,090, is 38% of the parent compound. The pharmacological properties of DOV 315,090 were assessed using radioligand binding studies and two-electrode voltage clamp electrophysiology. We report that DOV 315,090 possesses modulatory activity at GABAA-Rs, but that its selectivity profile is similar to that of ocinaplon. CONCLUSION: These findings imply that DOV 315,090 could contribute to the action of ocinaplon in vivo, but that the anxioselective properties of ocinaplon cannot be readily explained by a subtype selective effect/action of DOV 315,090. Further inquiry is required to identify the extent to which different subtypes are involved in the anxiolytic and other pharmacological effects of GABAA-R modulators.

The triple uptake inhibitor (1R,5S)-(+)-1-(3,4-dichlorophenyl)-3-azabicyclo[3.1.0] hexane hydrochloride (DOV 21947) reduces body weight and plasma triglycerides in rodent models of diet-induced obesity.

Selective inhibitors of biogenic amine (e.g., serotonin, norepinephrine, and dopamine) uptake exhibit varying degrees of safety and efficacy as antiobesity agents. Moreover, preclinical findings suggest that the combined inhibition of monoamine neurotransmitter transporters synergistically enhances antiobesity activity. (1R,5S)-(+)-1-(3,4-Dichlorophenyl)-3-azabicyclo-[3.1.0] hexane hydrochloride (DOV 21947) inhibits norepinephrine, 5-hydroxytryptamine, and dopamine uptake, and it reduces body weight in rodent models of diet-induced obesity (DIO). DIO rats treated orally with DOV 21947 for 1 to 24 days showed significantly lower body weights than vehicle-treated DIO rats. The decrease in body weight resulted specifically from a loss of retroperitoneal and mesenteric depots of white adipose tissue. DOV 21947 also reduced daily food intake in DIO rats, but consumption returned to control levels after 11 days of treatment. With the exception of a decrease in triglyceride levels, blood chemistry was unaltered after 24 days of DOV 21947 treatments. DOV 21947 had no effect on motor activity. Although DOV 21947 increased respiratory rate and decreased the tidal volume of normal rats, it did not alter the minute volume. In addition, DOV 21947 did not significantly affect blood pressure, heart rate, electrocardiographic indices or body temperature in telemeterized dogs. However, it caused a sustained, but reversible reduction in the rate of body weight gain for as long as 6 months in normal rats, and for up to 1 year in normal dogs. In summary, DOV 21947 is effective in causing a sustained and selective reduction in fat content and triglyceride levels in animal models of obesity without significantly altering vital organ function.

Induction of serine racemase expression and D-serine release from microglia by secreted amyloid precursor protein (sAPP).

Alzheimer's disease (AD) involves neuronal loss and reduction of synaptic density in specific brain region. Some of the neuronal deaths are associated with excitotoxicity. We previously reported that amyloid beta-peptide (Abeta) induced release of N-methyl-D-aspartate receptor (NMDA-R) co-agonists, including glutamate and D-serine. The induction of D-serine production by Abeta involves transcriptional and/or translational regulation of serine racemase gene. Similarly, we report here that conditioned medium from microglia treated with secreted amyloid precursor protein (sAPP) contained elevated levels of D-serine. In microglia, sAPP increased the steady-state dimeric protein level of serine racemase. Promoter-reporter and mRNA analyses suggested that serine racemase is transcriptionally induced by sAPP. These data extend the link between excitotoxicity and neuroinflammation. D-serine may cooperate with glutamate to link neuroinflammation with excitotoxicity, suggesting a pathogenic mechanism applicable to neuronal death in AD and other neurodegenerative diseases.

Triple reuptake inhibitors ("broad spectrum" antidepressants).

The majority of antidepressants in current use inhibit the uptake of serotonin and/or norepinephrine. Drugs inhibiting the uptake of serotonin, norepinephrine and dopamine (triple reuptake inhibitors) may offer therapeutic advantages compared to single and/or dual reuptake inhibitors. This review provides a rationale for developing this class of compound and describes the results of preclinical and clinical studies with a family of triple reuptake inhibitors.

Characterization of the antinociceptive actions of bicifadine in models of acute, persistent, and chronic pain.

Bicifadine (1-p-tolyl-3-azabicyclo[3.1.0]hexane) inhibits monoamine neurotransmitter uptake by recombinant human transporters in vitro with a relative potency of norepinephrine > serotonin > dopamine (approximately 1:2:17). This in vitro profile is supported by microdialysis studies in freely moving rats, where bicifadine (20 mg/kg i.p.) increased extrasynaptic norepinephrine and serotonin levels in the prefrontal cortex, norepinephrine levels in the locus coeruleus, and dopamine levels in the striatum. Orally administered bicifadine is an effective antinociceptive in several models of acute, persistent, and chronic pain. Bicifadine potently suppressed pain responses in both the Randall-Selitto and kaolin models of acute inflammatory pain and in the phenyl-p-quinone-induced and colonic distension models of persistent visceral pain. Unlike many transport inhibitors, bicifadine was potent and completely efficacious in both phases of the formalin test in both rats and mice. Bicifadine also normalized the nociceptive threshold in the complete Freund's adjuvant model of persistent inflammatory pain and suppressed mechanical and thermal hyperalgesia and mechanical allodynia in the spinal nerve ligation model of chronic neuropathic pain. Mechanical hyperalgesia was also reduced by bicifadine in the streptozotocin model of neuropathic pain. Administration of the D(2) receptor antagonist (-)-sulpiride reduced the effects of bicifadine in the mechanical hyperalgesia assessment in rats with spinal nerve ligations. These results indicate that bicifadine is a functional triple reuptake inhibitor with antinociceptive and antiallodynic activity in acute, persistent, and chronic pain models, with activation of dopaminergic pathways contributing to its antihyperalgesic actions.

The anxioselective agent 7-(2-chloropyridin-4-yl)pyrazolo-[1,5-a]-pyrimidin-3-yl](pyridin-2-yl)methanone (DOV 51892) is more efficacious than diazepam at enhancing GABA-gated currents at alpha1 subunit-containing GABAA receptors.

Studies using mice with point mutations of GABA(A) receptor alpha subunits suggest that the sedative and anxiolytic properties of 1,4-benzodiazepines are mediated, respectively, by GABA(A) receptors bearing the alpha(1) and alpha(2) subunits. This hypothesis predicts that a compound with high efficacy at GABA(A) receptors containing the alpha(1) subunit would produce sedation, whereas an agonist acting at alpha(2) subunit-containing receptors (with low or null efficacy at alpha(1)-containing receptors) would be anxioselective. Electrophysiological studies using recombinant GABA(A) receptors expressed in Xenopus oocytes indicate that maximal potentiation of GABA-stimulated currents by the pyrazolo-[1,5-a]-pyrimidine, DOV 51892, at alpha(1)beta(2)gamma(2S) constructs of the GABA(A) receptor was significantly higher (148%) than diazepam. In contrast, DOV 51892 was considerably less efficacious and/or potent than diazepam in enhancing GABA-stimulated currents mediated by constructs containing alpha(2), alpha(3), or alpha(5) subunits. In vivo, DOV 51892 increased punished responding in the Vogel conflict test, an effect blocked by flumazenil, and increased the percentage of time spent in the open arms of the elevated plus-maze. However, DOV 51892 had no consistent effects on motor function or muscle relaxation at doses more than 1 order of magnitude greater than the minimal effective anxiolytic dose. Although the mutant mouse data predict that the high-efficacy potentiation of GABA(A1a) receptor-mediated currents by DOV 51892 would be sedating, behavioral studies demonstrate that DOV 51892 is anxioselective, indicating that GABA potentiation mediated by alpha(1) subunit-containing GABA(A) receptors may be neither the sole mechanism nor highly predictive of the sedative properties of benzodiazepine recognition site modulators.

Pharmacological profile of the "triple" monoamine neurotransmitter uptake inhibitor, DOV 102,677.

1. The molecular and behavioral pharmacology of DOV 102,677 is characterized. 2. This characterization was performed using radioligand binding and neurotransmitter uptake assays targeting the monoamine neurotransmitter receptors. In addition, the effects of DOV 102,677 on extracellular neurotransmitter levels were investigated using in vivo microdialysis. Finally, the effects of DOV 102,677 in the forced swim test, locomotor function, and response to prepulse inhibition was investigated.3. DOV 102,677 is a novel, "triple" uptake inhibitor that suppresses [(3)H]dopamine (DA), [(3)H]norepinephrine (NE) and [(3)H]serotonin (5-HT) uptake by recombinant human transporters with IC(50) values of 129, 103 and 133 nM, respectively. Radioligand binding to the dopamine (DAT), norepinephrine (NET), and serotonin (SERT) transporters is inhibited with k (i) values of 222, 1030, and 740 nM, respectively. DOV 102,677 (20 mg/kg IP) increased extracellular levels of DA and 5-HT in the prefrontal cortex to 320 and 280% above baseline 100 min after administration. DA levels were stably increased for the duration (240 min) of the study, but serotonin levels declined to baseline by 200 min after administration. NE levels increased linearly to a maximum of 348% at 240 min post-dosing. Consistent with these increases in NE levels, the density of beta-adrenoceptors was selectively decreased in the cortex of rats treated with DOV 102,677 (20 mg/kg per day, PO, 35 days). 4. DOV 102,677 dose-dependently reduced the amount of time spent immobile by rats in the forced swim test, a model predictive of antidepressant activity, with a minimum effective dose (MED) of 20 mg/kg and a maximal efficacy comparable to imipramine. This decrease in immobility time did not appear to result from increased motor activity. Further, DOV 102,677 was as effective as methylphenidate in reducing the amplitude of the startle response in juvenile mice, without notably altering motor activity. 5. In summary, DOV 102,677 is an orally active, "balanced" inhibitor of DAT, NET and SERT with therapeutic versatility in treating neuropsychiatric disorders beyond depression.

M2 muscarinic acetylcholine receptor knock-out mice show deficits in behavioral flexibility, working memory, and hippocampal plasticity.

Muscarinic acetylcholine receptors are known to play key roles in facilitating cognitive processes. However, the specific roles of the individual muscarinic receptor subtypes (M1-M5) in learning and memory are not well understood at present. In the present study, we used wild-type (M2+/+) and M2 receptor-deficient (M2-/-) mice to examine the potential role of M2 receptors in learning and memory and hippocampal synaptic plasticity. M2-/- mice showed significant deficits in behavioral flexibility and working memory in the Barnes circular maze and the T-maze delayed alternation tests, respectively. The behavioral deficits of M2-/- mice were associated with profound changes in neuronal plasticity studied at the Schaffer-CA1 synapse of hippocampal slices. Strikingly, short-term potentiation (STP) was abolished, and long-term potentiation (LTP) was drastically reduced after high-frequency stimulation of M2-/- hippocampi. Treatment of M2-/- hippocampal slices with the GABA(A) receptor antagonist, bicuculline, restored STP and significantly increased LTP. Whole-cell recordings from CA1 pyramidal cells demonstrated a much stronger disinhibition of GABAergic than glutamatergic transmission in M2-/- hippocampi, which was particularly prominent during stimulus trains. Increased strength of GABAergic inhibition is thus a likely mechanism underlying the impaired synaptic plasticity observed with M2-/- hippocampi. Moreover, the persistent enhancement of excitatory synaptic transmission in CA1 pyramidal cells induced by the transient application of a low concentration of a muscarinic agonist (referred to as LTP(m)) was totally abolished in M2-/- mice. Because impaired muscarinic cholinergic neurotransmission is associated with Alzheimer's disease and normal aging processes, these findings should be of considerable therapeutic relevance.

Anxioselective anxiolytics: can less be more?

Benzodiazepines remain widely used for the treatment of anxiety disorders despite a side-effect profile that includes sedation, myorelaxation, amnesia, and ataxia, and the potential for abuse. gamma-Aminobutyric acid(A) (GABA(A)) receptor partial agonists, subtype-selective agents, and compounds combining both of these features are being developed in an attempt to achieve benzodiazepine-like efficacy without these potentially limiting side effects. This article reviews the nonclinical and clinical studies of "anxioselective" anxiolytics that target GABA(A) receptors and discusses potential mechanisms subserving an anxioselective profile.