Evaluation of the safety, pharmacokinetics and pharmacodynamic effects of subcutaneously administered PT-141, a melanocortin receptor agonist, in healthy male subjects and in patients with an inadequate response to Viagra.

PT-141, a cyclic heptapeptide melanocortin analog, was evaluated following subcutaneous administration to healthy male subjects and to patients with erectile dysfunction (ED) who report an inadequate response to Viagra. An inadequate response was defined for this study by patient report indicating that achievement of an erection suitable for vaginal penetration occurred < or =50% of the time while taking 100 mg Viagra. Erectile responses were assessed by RigiScan in healthy subjects in the absence of visual sexual stimulation (VSS) and in ED patients in the presence of VSS. Doses ranging from 0.3 to 10 mg were administered to healthy male subjects, resulting in a statistically significant erectile response at doses greater than 1.0 mg. ED patients were treated with placebo, 4 or 6 mg PT-141 in a crossover design in the presence of VSS. The erectile response induced by PT-141 was statistically significant at both doses. PT-141 was safe and well tolerated in both studies. The erectogenic potential of PT-141, its tolerability profile and its ability to cause significant erections in patients who do not have an adequate response to a PDE5 inhibitor suggest that PT-141 may provide an alternative treatment for ED with a potentially broad patient base.

Double-blind, placebo-controlled evaluation of the safety, pharmacokinetic properties and pharmacodynamic effects of intranasal PT-141, a melanocortin receptor agonist, in healthy males and patients with mild-to-moderate erectile dysfunction.

PT-141, a cyclic heptapeptide melanocortin analog, was evaluated following intranasal administration in healthy male subjects and in Viagra-responsive erectile dysfunction (ED) patients. Erectile response was assessed by RigiScan trade mark in healthy subjects without visual sexual stimulation (VSS) and in Viagra-responsive ED patients with VSS. In healthy subjects, mean C(max) and AUC((0-t)) increased in a dose-dependent manner. Median T(max) was 0.50 h and mean t(1/2) ranged from 1.85 to 2.09 h. In both studies, an erectile response induced by PT-141 administration was statistically significant, compared to placebo, at doses greater than 7 mg, with the onset of the first erection occurring in approximately 30 min. PT-141 was safely administered and well tolerated in both studies. A maximum-tolerated dose was not identified. Flushing and nausea were the most common adverse events reported in both studies and no clinically significant changes in vital signs, laboratory tests, ECGs, or physical exams were observed. Based upon its erectogenic potential and tolerability profile, PT-141 is a promising candidate for further evaluation as a treatment for male ED.

PT-141: a melanocortin agonist for the treatment of sexual dysfunction.

PT-141, a synthetic peptide analogue of alpha-MSH, is an agonist at melanocortin receptors including the MC3R and MC4R, which are expressed primarily in the central nervous system. Administration of PT-141 to rats and nonhuman primates results in penile erections. Systemic administration of PT-141 to rats activates neurons in the hypothalamus as shown by an increase in c-Fos immunoreactivity. Neurons in the same region of the central nervous system take up pseudorabies virus injected into the corpus cavernosum of the rat penis. Administration of PT-141 to normal men and to patients with erectile dysfunction resulted in a rapid dose-dependent increase in erectile activity. The results suggest that PT-141 holds promise as a new treatment for sexual dysfunction.

Targeting acute ischemic stroke with a calcium-sensitive opener of maxi-K potassium channels.

During ischemic stroke, neurons at risk are exposed to pathologically high levels of intracellular calcium (Ca++), initiating a fatal biochemical cascade. To protect these neurons, we have developed openers of large-conductance, Ca++-activated (maxi-K or BK) potassium channels, thereby augmenting an endogenous mechanism for regulating Ca++ entry and membrane potential. The novel fluoro-oxindoles BMS-204352 and racemic compound 1 are potent, effective and uniquely Ca++-sensitive openers of maxi-K channels. In rat models of permanent large-vessel stroke, BMS-204352 provided significant levels of cortical neuroprotection when administered two hours after the onset of occlusion, but had no effects on blood pressure or cerebral blood flow. This novel approach may restrict Ca++ entry in neurons at risk while having minimal side effects.

Common to both academia and industry: the challenge of discovery. An interview with Perry Molinoff.

Perry Molinoff recognizes the distinctions between basic and applied science, between academic and industrial research, and between the preclinical and clinical realities of drug development. But he generally discusses these categories in fluid, practical terms, having throughout his career crossed the lines of distinction that have sometimes been rather heavily drawn among pharmacologists. As a third-year medical student at Harvard, he decided "to take a year off" to conduct laboratory research. After receiving his MD and pursuing further clinical and postdoctoral work, he enjoyed an academic career that included fourteen years as the A.N. Richards Professor and Chair of Pharmacology at the University of Pennsylvania School of Medicine. He has just completed six years as Vice President of Neuroscience and Genitourinary Drug Discovery for Bristol-Myers Squibb and will soon return to teaching, in the Departments of Psychiatry and Pharmacology at Yale University. Referring to himself as either pharmacologist or neuroscientist, depending on context, he has made fundamental discoveries in receptor biology, has overseen the discovery and development of drugs and their subsequent clinical trials, and has mentored a host of pharmacologists and neuroscientists who themselves have established careers in industry and academia. The pursuit of discovery as its own reward emerges as a theme that has marked his professional life (and is perhaps reflected also in the images displayed in his office of the Himalayan mountains, photographed by Molinoff himself from the Everest base camp last year).

Regulation of levels of 5-HT2A receptor mRNA.

We have been able to identify two separate pathways present in P11 cells that can increase the levels and stability of 5-HT2A receptor mRNA. One pathway is activated following exposure of cells to serotonin and is dependent upon activation of a PKC isoform that is susceptible to downregulation by a 24-h pretreatment with PMA; another pathway is activated following treatment of cells with a calcium ionophore. This pathway is not dependent on activation of PKC isoforms that can be downregulated by a 24-h treatment with phorbol ester. Such heterologous regulation of levels of 5-HT2A receptor mRNA may be important in understanding in vivo events where multiple factors contribute to the modulation of levels of cell surface receptors.

5-hydroxytryptamine1A receptor-mediated increases in receptor expression and activation of nuclear factor-kappaB in transfected Chinese hamster ovary cells.

The regulation in expression of human 5-hydroxytryptamine1A (5-HT1A) receptors by agonists and antagonists was studied in a stable transfected Chinese hamster ovary cell line expressing the human 5-HT1A receptor. Receptor density and affinity were measured with [125I]4-(2'-methoxyphenyl)-1-[2'-[N-(2'-pyridinyl)-p-iodobenzamido ]ethyl]piperazine ([125I]p-MPPI), a selective antagonist of 5-HT1A receptors. Treatment of Chinese hamster ovary cells with serotonin or the selective agonist (+/-)-8-hydroxy-N,N-dipropyl-2-aminotetralin stimulated a 2.5-fold increase in receptor density. The antagonists 4-(2'-methoxyphenyl)-1-[2'-[N-(2'-pyridinyl)-p-iodobenzamidoethyl] piperazine, (-)-(S)-pindolol, and spiperone also stimulated up-regulation of receptor expression. Agonist- and antagonist-stimulated up-regulations of receptor expression were mechanistically different. The effect of agonists was inhibited by pertussis toxin, actinomycin D, and cycloheximide. Antagonist-stimulated up-regulation was inhibited by cycloheximide, only partially inhibited by actinomycin D, and not inhibited by pertussis toxin. In the course of identifying potential pathways for coupling of the receptor to activation of transcription, we demonstrated that agonists activate the transcription regulatory factor nuclear factor-kappaB (NF-kappaB). Agonists were found to stimulate degradation of the inhibitory subunit, IkappaB alpha, and to increase the activity of a NF-kappaB-dependent CAT reporter gene. In contrast, the antagonist 4-(2'-methoxyphenyl)-1-[2'-[N-(2'-pyridinyl)-p-iodobenzamidoethyl] piperazine neither elicited degradation of Ikappa-B alpha nor increased reporter activity. Our data suggest that expression of 5-HT1A receptors can be regulated by both agonists and antagonists and that the agonist but not antagonist stimulation occurs concomitantly with activation of NF-kappaB.

Use of subunit-specific antisense oligodeoxynucleotides to define developmental changes in the properties of N-methyl-D-aspartate receptors.

Antisense oligodeoxynucleotides were used to determine whether alterations in the expression of N-methyl-D-aspartate (NMDA) receptor subunit mRNA are responsible for developmental changes in the sensitivity of receptors to agonists and antagonists. Xenopus laevis oocytes were injected with mRNA prepared from neonatal and adult rat cerebral cortex, and the effects of agonists and antagonists were determined under voltage-clamp conditions. Glycine-site antagonists like 7-chlorokynurenate and glutamate-site antagonists like CGP-39653 were more potent at NMDA receptors expressed from mRNA from adult rat cerebral cortex than those expressed from mRNA from 1-day-old rat. NMDA receptors from 1-day-old rat cerebral cortex were more sensitive to activation by glycine than were receptors from adult rat cerebral cortex. 7-Chlorokynurenate and CGP-39653 were more potent inhibitors of responses seen with heteromeric NR1/NR2A receptors than with NR1/ NR2B receptors. Conversely, heteromeric NR1/NR2B receptors were more sensitive to activation by glycine than were NR1/NR2A receptors. We previously described a delay in the expression of the NR2A subunit in developing rat brain. Anti-sense oligodeoxynucleotides were used to determine whether the delayed expression of the NR2A subunit underlies changes in pharmacological properties observed during development. The properties of receptors seen when adult brain mRNA was coinjected with antisense oligodeoxynucleotides against the NR2A subunit were similar to those found in receptors from 1-day-old rat brain. These data suggest that changes in the sensitivity of NMDA receptors to antagonists and to glycine seen during development are a result of alterations in the expression of different species of NR2 subunit mRNA.

Differential coupling of rat D2 dopamine receptor isoforms expressed in Spodoptera frugiperda insect cells.

By using a baculovirus expression system, the two isoforms of the rat D2 dopamine receptor were expressed at densities ranging up to 15 pmol/mg of protein. D2L and D2S dopamine receptors expressed in aline of Spodoptera frugiperda (Sf9) insect cells Sf9cells, displayed high affinity for the antagonists spiroperidol and (+)-butaclamol and the agonist N-propylnorapomorphine. Antisera raised against the D2 receptor immunoprecipitated binding sites for a radiolabeled D2 antagonist from solubilized extracts of infected Sf9cells. In immunoblots of Sf9cells infected with recombinant D2 baculovirus, these antisera recognized a major species of protein of approximately 46 kDa. Photoaffinity-labeling of infected Sf9cells using N-(p-azido-m-[125I]iodophenethyl)spiperone also identified a protein of this size, suggesting that D2 receptors expressed in Sf9cells are largely unglycosylated. In cells expressing receptors at a density greater than 1 pmol/mg, GTP-sensitive, high-affinity binding of agonists was not detected in studies of the inhibition of the binding of a radiolabeled D2 antagonist. When expression levels were under 1 pmol/mg, the binding of agonists was sensitive to the addition of guanine nucleotides, indicating that D2 receptors were coupled to endogenous G proteins. Endogenous G proteins enable both isoforms of D2 receptors to couple to the inhibition of adenylyl cyclase activity. The high-affinity state of the D2 receptor was directly measured using a radiolabeled agonist. Although the density of receptors increased with longer times after infection, the density of high-affinity sites reached a maximum of approximately 40 fmol/mg 30 to 36 hr after infection. Coexpression of D2 receptors and G protein subunits in Sf9cells dramatically increased the density of high-affinity sites, whereas the total density of receptors was unchanged, confirming that D2 receptors in Sf9 cells can exist in the high-affinity-coupled state, but that appropriate G proteins are expressed at relatively low levels. The density of D2S receptors converted to a coupled, agonist-preferring state when coexpressed with G proteins subunits (alpha i1, beta 1 and gamma 2) was 5 times greater than that of D2L receptors expressed under the same conditions, consistent with the hypothesis that D2 dopamine receptor isoforms differentially couple to alpha i1.

Regulation of 5-HT2A receptor mRNA in P11 cells.

P11 cells were used as an in vitro model system to explore effects of exposure to 5-HT on levels of mRNA encoding 5-HT2A receptors. Exposure of cells to the agonist 5-HT resulted in a doubling of receptor mRNA levels. mRNA levels returned to control levels within 8-16 h. The stability of receptor mRNA transcripts was transiently increased, suggesting that a post-transcriptional process was responsible for the up-regulation of receptor mRNA. The 5-HT-induced increase in Levels on 5-HT2A receptor mRNA did not require de novo protein synthesis since the increase was not affected by prior treatment with the protein synthesis inhibitor cycloheximide. Results of studies in which the activity of protein kinase C was either increased with PMA or antagonized with bisindolylmaleimide indicated that protein phosphorylation was essential for increasing levels of 5-HT2A receptor mRNA. These findings suggest that PKC-dependent post-transcriptional and post-translational processes participate in regulating 5-HT2A receptor mRNA expression.

Agonists and antagonists differentially regulate the high affinity state of the D2L receptor in human embryonic kidney 293 cells.

Studies with radiolabeled antagonists have revealed that both agonists and antagonists induce up-regulation of D2 dopamine receptors in cells transfected to express D2L or D2S receptors. The regulation induced by agonists, but not antagonists, was synergistic with cAMP analogues, and differences in the time courses of the effects of agonists and antagonists have been observed. These findings have been extended by using a radiolabeled agonist to investigate agonist- and antagonist-induced regulation of the high affinity state of the D2L dopamine receptor in transfected HEK 293 cells. Exposure to agonists decreased the proportion of receptors in the high affinity, agonist-preferring state. Exposure to antagonists, however, led to an increase in the density of receptors with a high affinity for agonists. The effects of both agonists and antagonists on the agonist-preferring receptors occurred without a lag and were time and dose dependent. Inhibition of forskolin-stimulated cAMP accumulation by agonists was not affected by exposure of the cells to the antagonist (-)-sulpiride. Desensitization was seen after exposing cells to the agonist quinpirole for 1.5 hr, suggesting that the rapid loss of high affinity binding sites represents an uncoupling of the receptor from the G protein that mediates inhibition of adenylyl cyclase. Pretreatment of cells with the protein synthesis inhibitor cycloheximide did not block the quinpirole-induced loss of receptors with a high affinity for agonists. The effect of (-)-sulpiride on high affinity binding sites was blocked by cycloheximide, but only after incubation of cells for sufficient time to induce an increase in the total number of receptors. After incubation of cells with (-)-sulpiride for a short time, the increase in the number of receptors with a high affinity for agonists was unaffected by cycloheximide. These results suggest that the increase in agonist binding after brief exposure to an antagonist is due to interactions of the receptor with one or more G proteins that are not coupled to inhibition of adenylyl cyclase, whereas the increase in agonist binding at later time points is associated with the antagonist-induced up-regulation.

Expression of the human 5-hydroxytryptamine1A receptor in Sf9 cells. Reconstitution of a coupled phenotype by co-expression of mammalian G protein subunits.

The possibility that Spodoptera frugiperda (Sf9) cells can provide an intact cell setting for reconstitution of the human 5-hydroxytryptamine1A (5-HT1A) receptor with mammalian G protein subunits was explored. The 5-HT1A receptor was found to assume an uncoupled phenotype when expressed alone in Sf9 cells at relatively high levels (5-34 pmol of receptor/mg of membrane protein), i.e. agonist-binding to the receptor was characterized by a relatively high Kd and an insensitivity to GTP. Co-expression of the receptor with members of the alpha i "family" together with various combinations of beta 1 and gamma subunits increased the affinity for agonists to that observed for the coupled form of receptor in mammalian cells, concomitant with conferrance of guanosine 5'-(beta,gamma-imino)triphosphate sensitivity. The agonists employed were [3H]8-hydroxy-N,N-dipropyl-2-aminotetralin ([3H]8-OH-DPAT) and [125I]R(+)-trans-8-hydroxy-2-[N-n-propyl-N-(3'-iodo-2'-propenyl) amino]tetralin ([125I]8-OH-PIPAT). The binding of an antagonist, [125I]4-(2'-methoxyphenyl)-1-[2'-[N-(2"- pyridinyl)-p-iodobenzamido]ethyl]piperazine ([125I]p-MPPI), was unaffected by co-expression of G protein subunits. Both alpha and beta gamma subunits were required for optimal coupling. No differences were evident among alpha i1, alpha i2, alpha i3, alpha o, and alpha z when expressed with beta 1 gamma 2 in this regard, nor among most permutations of beta 1 gamma subunits when expressed with alpha i1 (beta 1 gamma 2 approximately beta 1 gamma 3 approximately beta 1 gamma 5 > beta 1 gamma 1). Alpha s and alpha q expressed with beta 1 gamma 2 did not participate in coupling. These data support the conclusion that normal interactions between a mammalian receptor and a select array of G proteins can be established in intact Sf9 cells, and extend previous observations of 5-HT1A receptor coupling to G(o) and the pertussis toxin-insensitive G protein Gz.

Lack of discrimination by agonists for D2 and D3 dopamine receptors.

The affinities of D3 dopamine receptors for antagonists are similar to those of D2 receptors. D3 receptors have been reported, however, to have affinities nearly 100-fold higher than those of D2 receptors for some agonists, including (+/-)-7-hydroxy-n,n-dipropyl-aminotetralin (7-OH-DPAT) and quinpirole. This has led to the use of these agonists to try to identify functional responses mediated by D3 receptors in vivo. However, D2 receptors exist in multiple states having high and low affinities for agonists. The G protein-coupled state of D2 receptors is believed to be the functional state of these receptors. When receptors were labeled with the D2 receptor antagonist [125I]-(S)-3-iodo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5,6- dimethoxysalicylamide ([125I]-NCQ-298) under conditions that promote uncoupling of receptors from G proteins, the affinities of D3 receptors were approximately 130-fold higher than those of D2 receptors for 7-OH-DPAT and quinpirole. When receptors were labeled with the D2 receptor agonist [125I]-(R)trans-7-hydroxy-2-[N-propyl-N-(3'-iodo-2'- propenyl)-amino]tetralin ([125I]-7-OH-PIPAT) under conditions that favor interactions of receptors with G proteins, the affinities of D3 receptors were less than sevenfold higher than the affinities of D2 receptors for the same drugs. Similarly, small differences in the affinities of D2 and D3 receptors for other agonists were seen when receptors were labeled with [125I]-7-OH-PIPAT. These data demonstrate that putative D3 receptor-selective agonists also interact with a high-affinity, G protein-coupled state of D2 receptors. The similarities in affinities of the agonist-preferring state of D2 and D3 receptors means that currently available agonists cannot be used to discriminate between behavioral effects mediated by D2 and D3 receptors.

Expression of mRNAs encoding subunits of the NMDA receptor in developing rat brain.

Developmental changes in the levels of N-methyl-D-aspartate (NMDA) receptor subunit mRNAs were identified in rat brain using solution hybridization/RNase protection assays. Pronounced increases in the levels of mRNAs encoding NR1 and NR2A were seen in the cerebral cortex, hippocampus, and cerebellum between postnatal days 7 and 20. In cortex and hippocampus, the expression of NR2B mRNA was high in neonatal rats and remained relatively constant over time. In contrast, in cerebellum, the level of NR2B mRNA was highest at postnatal day 1 and declined to undetectable levels by postnatal day 28. NR2C mRNA was not detectable in cerebellum before postnatal day 11, after which it increased to reach adult levels by postnatal day 28. In cortex, the expression of NR2A and NR2B mRNAs corresponds to the previously described developmental profile of NMDA receptor subtypes having low and high affinities for ifenprodil, i.e., a delayed expression of NR2A correlating with the late expression of low-affinity ifenprodil sites. In cortex and hippocampus, the predominant splice variants of NR1 were those without the 5' insert and with or without both 3' inserts. In cerebellum, however, the major NR1 variants were those containing the 5' insert and lacking both 3' inserts. The results show that the expression of NR1 splice variants and NR2 subunits is differentially regulated in various brain regions during development. Changes in subunit expression are likely to underlie some of the changes in the functional and pharmacological properties of NMDA receptors that occur during development.

Regulation of mRNA encoding 5-HT2A receptors in P11 cells through a post-transcriptional mechanism requiring activation of protein kinase C.

Exposure of P11 cells to serotonin (5-HT) resulted in a transient increase in levels of 5-HT2A receptor mRNA. Exposure to 5-HT for as short a time as 1 min was sufficient to trigger a delayed increase in receptor mRNA. 5-HT-induced increases in receptor mRNA levels were not antagonized by the protein synthesis inhibitor cycloheximide. The increase in receptor mRNA levels was accompanied by a transient increase in the half-life of receptor mRNA; the rate of transcription of receptor mRNA was unchanged. Submaximal stimulation of phosphinositide hydrolysis by partial agonists or 6-fluoronorepinephrine, an alpha 1-adrenergic receptor agonist, also increased receptor mRNA levels. Exposure to phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, mimicked these effects, whereas the protein kinase C inhibitor bisindolylmaleimide antagonized the effects of both 5-HT and PMA. When agonist-promoted increases in receptor mRNA were prevented, the rate of agonist-induced down-regulation was accelerated. These data suggest that levels of 5-HT2A receptor mRNA are regulated by phospholipase C-coupled receptors via a protein kinase C-dependent, post-transcriptional mechanism and indicate that agonist-promoted increases in levels of 5-HT2A receptor mRNA modulate receptor expression.

Mechanisms of up-regulation of D2L dopamine receptors by agonists and antagonists in transfected HEK-293 cells.

Mechanisms underlying agonist- and antagonist-induced up-regulation in HEK-293 cells transfected to express D2 dopamine receptors were investigated. We have reported previously that exposure of cells to agonists and antagonists led to an increase in the density of receptors. The time course of up-regulation on exposure to (-)-sulpiride, a D2 dopamine-selective antagonist, was measured. A lag in the effect of antagonists not seen with up-regulation induced by exposure to agonists was observed. Effects of N-propylnorapomorphine were synergistic with those of cyclic AMP analogs, however, synergistic effects between (-)-sulpiride and cyclic AMP analogs were not observed. These findings suggest that separate mechanisms may be involved in agonist- and antagonist-induced up-regulation. Changes in mRNA levels did not appear to account for the increase in receptors after agonist or antagonist treatment. Results of experiments with cycloheximide, a protein-synthesis inhibitor, suggest that increased protein synthesis, and not decreased protein degradation, is responsible for up-regulation by both NPA and (-)-sulpiride. Studies monitoring receptor recovery after treatment with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, an irreversible alkylating agent, suggest that rates of receptor incorporation into membranes are increased after treatment with either an agonist or an antagonist.

Characterization of binding sites for [125I]R(+)trans-7-OH-PIPAT in rat brain.

Binding characteristics of a novel radioiodinated ligand, [125I]R(+)trans-7-hydroxy-2-(N-n-propyl-N-3'-iodo-2'-propenyl) aminotetralin ([125I]R(+)trans-7-OH-PIPAT), were evaluated using homogenate binding and autoradiographic techniques in rat brain. [125I]R(+)trans-7-OH-PIPAT bound to sites (dopamine receptors) in homogenates of rat basal forebrain (including caudate putamen, nucleus accumbens and olfactory tubercle) with a high affinity (Kd = 0.42 nM). A majority (70%) of the sites labeled by [125I]R(+)trans-7-OH-PIPAT in basal forebrain were GTP-sensitive. In rat hippocampal homogenates, specific and saturable binding of [125I]R(+)trans-7-OH-PIPAT to 5-HT1A receptors, with a Kd value of 1.4 nM and a Bmax value of 210 fmol/mg protein, was observed. Binding of [125I]R(+)trans-7-OH-PIPAT to sigma sites was also demonstrated in rat cerebellar homogenates. In the presence of GTP (to inhibit binding to D2 and 5-HT1A receptors) and DTG (to inhibit binding to sigma sites), dopamine D3 receptors could be selectively labeled with [125I]R(+)trans-7-OH-PIPAT. [125I]R(+)trans-7-OH-PIPAT offers several unique advantages, including high specific activity and high affinity binding, which make it an excellent probe for the investigation and characterization of the distribution of dopamine D3 receptors.