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.

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.

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.

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.

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.

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.

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.

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.

Multiple muscarinic acetylcholine receptor subtypes modulate striatal dopamine release, as studied with M1-M5 muscarinic receptor knock-out mice.

A proper balance between striatal muscarinic cholinergic and dopaminergic neurotransmission is required for coordinated locomotor control. Activation of striatal muscarinic acetylcholine receptors (mAChRs) is known to modulate striatal dopamine release. To identify the mAChR subtype(s) involved in this activity, we used genetically altered mice that lacked functional M1-M5 mAChRs [knock-out (KO) mice]. In superfused striatal slices from wild-type mice, the non-subtype-selective muscarinic agonist oxotremorine led to concentration-dependent increases in potassium-stimulated [3H]dopamine release (by up to 60%). The lack of M1 or M2 receptors had no significant effect on the magnitude of these responses. Strikingly, oxotremorine-mediated potentiation of stimulated striatal [3H]dopamine release was abolished in M4 receptor KO mice, significantly increased in M3 receptor-deficient mice, and significantly reduced (but not abolished) in M5 receptor KO mice. Additional release studies performed in the presence of tetrodotoxin suggested that the dopamine release-stimulating M4 receptors are probably located on neuronal cell bodies, but that the release-facilitating M5 and the release-inhibiting M3 receptors are likely to be located on nerve terminals. Studies with the GABA(A) receptor blocker bicuculline methochloride suggested that M3 and M4 receptors mediate their dopamine release-modulatory effects via facilitation or inhibition, respectively, of striatal GABA release. These results provide unambiguous evidence that multiple mAChR subtypes are involved in the regulation of striatal dopamine release. These findings should contribute to a better understanding of the important functional roles that the muscarinic cholinergic system plays in striatal function.

Characterization of central inhibitory muscarinic autoreceptors by the use of muscarinic acetylcholine receptor knock-out mice.

Forebrain muscarinic acetylcholine (ACh) receptors (mAChRs; M1-M5) are predicted to play important roles in many fundamental central functions, including higher cognitive processes and modulation of extrapyramidal motor activity. Synaptic ACh levels are known to be regulated by the activity of presynaptic muscarinic autoreceptors mediating inhibition of ACh release. Primarily because of the use of ligands with limited receptor subtype selectivity, classical pharmacological studies have led to conflicting results regarding the identity of the mAChR subtypes mediating this activity in different areas of the brain. To investigate the molecular identity of hippocampal, cortical, and striatal inhibitory muscarinic autoreceptors in a more direct manner, we used genetically altered mice lacking functional M2 and/or M4 mAChRs [knock-out (KO) mice]. After labeling of cellular ACh pools with [3H]choline, potassium-stimulated [3H]ACh release was measured in superfused brain slices, either in the absence or the presence of muscarinic drugs. The nonsubtype-selective muscarinic agonist, oxotremorine (0.1-10 microm), inhibited potassium-stimulated [3H]ACh release in hippocampal, cortical, and striatal slices prepared from wild-type mice by up to 80%. This activity was totally abolished in tissues prepared from M2-M4 receptor double KO mice. Strikingly, release studies with brain slices from M2 and M4 receptor single KO mice indicated that autoinhibition of ACh release is mediated primarily by the M2 receptor in hippocampus and cerebral cortex, but predominantly by the M4 receptor in the striatum. These results, together with additional receptor localization studies, support the novel concept that autoinhibition of ACh release involves different mAChRs in different regions of the brain.

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.

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.

Vivitrex, an injectable, extended-release formulation of naltrexone, provides pharmacokinetic and pharmacodynamic evidence of efficacy for 1 month in rats.

While oral naltrexone is effective in treating alcohol and opiate dependencies, poor patient adherence and widely fluctuating plasma levels limit its efficacy. To overcome these problems, an extended-release formulation of naltrexone (Vivitrex) was developed by encapsulating naltrexone into injectable, biodegradable polymer microspheres. Pharmacokinetic studies in rats demonstrated that this formulation produced stable, pharmacologically relevant plasma levels of naltrexone for approximately 1 month following either subcutaneous or intramuscular injections. While rats receiving placebo microspheres demonstrated a pronounced analgesic response to morphine in the hot-plate test, morphine analgesia was completely blocked in rats treated with extended-release naltrexone. This antagonism began on day 1 following administration and lasted for 28 days. Rats reinjected with extended-release naltrexone 34 days after the initial dose and tested for another 35 days showed consistent suppression of morphine analgesia for an additional 28 days. mu-Opioid receptor density, as measured by [(3)H]DAMGO autoradiography, increased up to two-fold following a single injection of extended-release naltrexone. Saturation binding assays using [(3)H]DAMGO showed changes in the midbrain and striatum at 1 week after extended-release naltrexone administration, and after 1 month in the neocortex. These receptor increases persisted for 2-4 weeks after dissipation of the morphine antagonist actions of naltrexone. These data suggest that therapeutically relevant plasma levels of naltrexone can be maintained using monthly injections of an extended-release microsphere formulation, and that changes in mu-opioid receptor density do not impact its efficacy in suppressing morphine-induced analgesia in the rat. Clinical trials of extended release naltrexone for treating alcohol and opiate dependency are currently ongoing.

Induction of serine racemase expression and D-serine release from microglia by amyloid beta-peptide.

BACKGROUND: Roles for excitotoxicity and inflammation in Alzheimer's disease have been hypothesized. Proinflammatory stimuli, including amyloid beta-peptide (Abeta), elicit a release of glutamate from microglia. We tested the possibility that a coagonist at the NMDA class of glutamate receptors, D-serine, could respond similarly. METHODS: Cultured microglial cells were exposed to Abeta. The culture medium was assayed for levels of D-serine by HPLC and for effects on calcium and survival on primary cultures of rat hippocampal neurons. Microglial cell lysates were examined for the levels of mRNA and protein for serine racemase, the enzyme that forms D-serine from L-serine. The racemase mRNA was also assayed in Alzheimer hippocampus and age-matched controls. A microglial cell line was transfected with a luciferase reporter construct driven by the putative regulatory region of human serine racemase. RESULTS: Conditioned medium from Abeta-treated microglia contained elevated levels of D-serine. Bioassays of hippocampal neurons with the microglia-conditioned medium indicated that Abeta elevated a NMDA receptor agonist that was sensitive to an antagonist of the D-serine/glycine site (5,7-dicholorokynurenic acid; DCKA) and to enzymatic degradation of D-amino acids by D-amino acid oxidase (DAAOx). In the microglia, Abeta elevated steady-state levels of dimeric serine racemase, the apparent active form of the enzyme. Promoter-reporter and mRNA analyses suggest that serine racemase is transcriptionally induced by Abeta. Finally, the levels of serine racemase mRNA were elevated in Alzheimer's disease hippocampus, relative to age-matched controls. CONCLUSIONS: These data suggest that Abeta could contribute to neurodegeneration through stimulating microglia to release cooperative excitatory amino acids, including D-serine.

Direct and indirect enhancement of GABAergic neurotransmission by ammonia: implications for the pathogenesis of hyperammonemic syndromes.

Experimental evidence indicates that ammonia causes neuroexcitation and seizures. This contrasts with the lethargy, confusion and other manifestations of global CNS depression commonly considered to be major components of hyperammonemic encephalopathies. Substantial data now indicates that ammonia can modulate GABAergic neurotransmission through direct and indirect mechanisms. This modulation consists of an enhancement of GABAergic neurotransmission at concentrations commonly encountered in hyperammonemic states and precedes the suppression of inhibitory neuronal function observed at higher (>1mM) ammonia concentrations. Not only is this increase in GABAergic neurotransmission consistent with the clinical picture of lethargy, ataxia and cognitive deficits associated with liver failure and congenital hyperammonemia, but it also provides a mechanism for testing new therapeutic modalities for the treatment of hyperammonemic encephalopathy.

Peripheral benzodiazepine receptors and mitochondrial function.

For over 20 years, numerous investigations have focused on elucidating the function of the peripheral benzodiazepine receptor (PBR). This relatively small protein (18kDa) arouses great interest because of its association with numerous biological functions, including the regulation of cellular proliferation, immunomodulation, porphyrin transport and heme biosynthesis, anion transport, regulation of steroidogenesis and apoptosis. Although the receptor was first identified as a binding site for the benzodiazepine, diazepam, in peripheral organ systems, the PBR was subsequently found to be distinct from the central benzodiazepine receptor (CBR) in terms of its pharmacological profile, structure, subcellular localization, tissue distribution and physiological functions. The PBR is widely expressed throughout the body, with high densities found in steroid-producing tissues. In contrast, its expression in the CNS is restricted to ependymal cells and glia. The benzodiazepine Ro5-4864 and the isoquinoline carboxamide PK11195 exhibit nanomolar affinity for the PBR, and are the archtypic pharmacological tools for characterizing the receptor and its function. Primary among these functions are its regulation of steroidogenesis and apoptosis, which reflect its mitochondrial localization and involvement in oxidative processes. This review will evaluate the basic pharmacology and molecular biology of the PBR, and highlight its role in regulating mitochondrial function, the mitochondrial transmembrane potential and its sensitivity to reactive oxygen species (ROS), and neurosteroid synthesis, processes relevant to the pathogenesis of a number of neurological and neuropsychiatric disorders.

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.

Novel insights into M5 muscarinic acetylcholine receptor function by the use of gene targeting technology.

Until recently, little was known about the possible physiological functions of the M(5) muscarinic acetylcholine receptor subtype, the last member of the muscarinic receptor family (M(1)-M(5)) to be cloned. To learn more about the potential physiological roles of this receptor subtype, we generated and analyzed M(5) receptor-deficient mice (M5 -/- mice). Strikingly, acetylcholine, a potent dilator of most vascular beds, virtually lost the ability to dilate cerebral arteries and arterioles in M5 -/- mice, suggesting that endothelial M(5) receptors mediate this activity in wild-type mice. This effect was specific for cerebral blood vessels, since acetylcholine-mediated dilation of extra-cerebral arteries remained fully intact in M5 -/- mice. In addition, in vitro neurotransmitter release experiments indicated that M(5) receptors located on dopaminergic nerve terminals play a role in facilitating muscarinic agonist-induced dopamine release in the striatum, consistent with the observation that the dopaminergic neurons innervating the striatum almost exclusively express the M(5) receptor subtype. We also found that the rewarding effects of morphine, the prototypical opiate analgesic, were substantially reduced in M5 -/- mice, as measured in the conditioned place preference paradigm. Furthermore, both the somatic and affective components of naloxone-induced morphine withdrawal symptoms were significantly attenuated in M5 -/- mice. It is likely that these behavioral deficits are caused by the lack of mesolimbic M(5) receptors, activation of which is known to stimulate dopamine release in the nucleus accumbens. These results convincingly demonstrate that the M(5) muscarinic receptor is involved in modulating several important pharmacological and behavioral functions. These findings may lead to novel therapeutic strategies for the treatment of drug addiction and certain cerebrovascular disorders.

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.

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).