• Design, synthesis and pharmacological evaluation of orphanin FQ(1-13)amidogen analogs

      Charoenchai, Laksana; Aldrich, Jane V. (2001)
      The novel orphanin (ORL1) receptor and its endogenous ligand orphanin FQ or nociceptin (OFQ/N) are similar to the opioid receptors and ligands, particularly the Kopioid receptor and dynorphin A (Dyn A). OFQ exhibits various physiological and pharmacological effects but the role of OFQ in nociception is not well understood. Thus OFQ analogs are useful as pharmacological tools to study the ORL1 receptor. One goal of this research was to prepare constrained analogs of OFQ(1-13)NH2 to explore the structural and conformational requirements of this peptide for receptor binding and activation. Initially linear D/L-Asn and D/L-Met OFQ(1-13)NH 2 analogs were synthesized to determine possible positions and linkage for the design of constrained OFQ(1-13)NH2 analogs. Other linear OFQ(1-13)NH2 analogs with substitutions of various amino acids at positions 2 to 7 (excluding position 4) were also prepared to examine the effects of different side chain functional groups on ligand-receptor interaction. Two series of cyclic peptides with a lactam linkage were prepared with the constraint incorporated into different regions of the parent peptide. Hybrid OFQ peptides were also designed by incorporating novel N-terminal sequences from Dyn A analogs into OFQ(1-13)NH2 to examine the effects on ORL1 receptor affinity versus the Dyn A analogs. The peptides were prepared by solid phase synthesis using the Fmoc (9-fluoronylmethoxycarbonyl)/tert-butyl synthetic protocol. The compounds were evaluated for their affinity and efficacy for cloned human orphanin receptor (hORL1) expressed in Chinese hamster ovary (CHO) cells using a radioligand binding and the [35S]GTPgammaS assays, respectively. Modification with a variety of L/D-amino acids in the middle of the peptide sequence resulted in high affinity analogs. [D-Ala7]OFQ(1-13)NH2 was a full agonist in the GTPgammaS assay, in contrast to the literature report' that [D-Ala7]OFQ is a partial agonist. Cyclization via a lactam linkage in the middle of the OFQ sequence resulted in analogs with high affinity (Ki = 0.27-7 nM) and high to moderate potency (EC50 = 1.6-157 nM) in the GTPgammaS assay. Two cyclic analogs exhibited higher affinity and potency than the parent peptide. Hybrid OFQ analogs which had novel N-terminal sequences from Dyn A analogs generally exhibited low or no binding affinity at the ORL1 receptor. Thus the structural requirements in the N-terminal sequence of OFQ and Dyn A are different. These structure-activity relationship (SAR) studies provide additional information on the structural features for consideration in the further design of novel OFQ ligands. The high affinity analogs described here, particularly the cyclic peptides, will be useful pharmacological tools to study the roles of the orphanin system.
    • The drug delivery factors that influence the tissue distribution and pharmacokinetics of a series of active antiepileptic agents, the enaminones

      Cox, Donna S.; Eddington, Natalie D. (2000)
      Early Central Nervous System (CNS) discovery generated new pharmaceuticals based on two properties of the compound, lipophilicity and molecular size. Almost all CNS pharmaceuticals presently in clinical trials possess both of these criteria and are capable of crossing the blood-brain barrier (BBB). Currently, modern methods of drug discovery utilize high throughput screening methods that may select molecules that lack both properties and will not undergo transport across the BBB. With the advent of in vitro cell culture models that mimic the BBB in vivo, researchers are now able to evaluate drug interactions at the BBB and to elucidate mechanisms at both the cellular and molecular levels pertinent for drug delivery. The utilization of both in vitro models for relatively rapid screening of permeability and related transport mechanisms, and in vivo models to assess drug pharmacokinetic distribution to the CNS provides a powerful assessment of drug delivery across the BBB to the CNS. Enaminone esters in the carbomethoxy series have previously been evaluated and shown to possess potent oral anticonvulsant activity in the mouse and rat. However, preliminary studies assessing enaminone analogs did not show expected correlations between relevant physiochemical parameters such as lipophilicity and BBB permeability. Therefore, in vitro models were utilized to assess factors associated with drug transport. An in vitro model of the BBB, Bovine Brain Microvessel Endothelial cells (BBMECs) were isolated and used to evaluated permeability and cellular mechanisms influencing enaminone transport. Results demonstrated that a multidrug resistant protein (MDR) influences enaminone permeability at the BBB. Further elucidation of possible mechanisms influencing enaminone distribution and pharmacokinetics were performed in a genetically altered mouse model [mdr1a/ b (--/--)] deficient in the expression of P-glycoprotein. Results comparing the brain distribution and partition coefficients in knockout mdr1a/b (--/--) versus wild type (+/+) counterparts demonstrated a higher accumulation of enaminones in brain tissue of knockout mice. Pharmacokinetic analysis of the tissue disposition of enaminones additionally demonstrated that the lack of P-glycoprotein in the lung and liver influence drug disposition in knockout animals. Lastly, a physiological based pharmacokinetic model was developed and found to be predictive of DM5 [methyl 4-[(4-chlorophenyl) amino]-6-methyl-2-oxocyclohex-3-en-1-oate] tissue distribution in mdr1a/b (--/--) knockout and wild type mice.
    • The evaluation of the metabolism of N-methylspiperone and its effect on kinetic and receptor binding parameter estimation

      Miller, Ann Kay; Young, David G. (1992)
      N-methylspiperone (NMSP) is a butyrophenone derivative that binds preferentially to dopamine D2 receptors in the brain of animals and man. This dissertation describes the development of a specific and sensitive HPLC assay for quantitation of NMSP in guinea pig plasma and brain. The metabolism of NMSP is studied in guinea pigs and rats after multiple intraperitoneal injections of NMSP. Three important metabolites found in guinea pig plasma and brain were identified as reduced NMSP, spiperone, and reduced spiperone. Only spiperone was found in rats. Furthermore, reduced NMSP was found to be a radiolabeled metabolite after single or multiple doses of {dollar}\sp3{dollar}H-NMSP and after a single dose of {dollar}\sp{lcub}11{rcub}{dollar}C-NMSP in guinea pigs. The plasma disposition of NMSP and {dollar}\sp3{dollar}H-NMSP in guinea pigs after a single intravenous dose was best described by a biexponential decline. The half-lives of NMSP distribution and elimination were 11.7 and 289 minutes, respectively. Reduced NMSP was found to follow a mono- or biexponential decline in plasma after a single intravenous dose. The mean elimination half-life was 54.7 minutes. The "donor-recipient" parameter estimation method was evaluated for usefulness in NMSP receptor binding studies. This method includes estimation of transfer rates of formation of a radiolabeled metabolite in the body and transfer of the metabolite into and out of the brain. Data was simulated for a radiolabeled parent compound and its radiolabeled metabolite in the plasma and in the two types of brain regions--those with and without specific binding of parent to the receptors. All parameters except the rate constant of metabolite elimination from the body were estimated within 1% of the true value. Parameters estimated by non-linear least squares regression analysis were found to be similar to those from the "donor-recipient" method. In evaluating the "donor-recipient" method when specific binding is assumed to be reversible, only K23 was poorly estimated. The "donor-recipient" method does provide accurate estimation of all parameters of interest in the receptor binding models determined in this analysis. However, K23, the rate of sequestration into the brain compartment, is only accurately estimated when it is truly represented by a first order process and K23 is zero. (Abstract shortened with permission of author.)
    • The in vitro and in vivo studies of the structure and functional regulation of the mu-opioid receptor

      Deng, Hong Bing; Wang, Jia Bei (1999)
      A prominent characteristic of opioid drugs is their ability to induce tolerance and dependence in human. The mu receptor phosphorylation and desensitization are important cellular mechanisms that may contribute to these neuroadaptive processes. The overall goal of the present research is to understand the molecular mechanisms of mu opioid receptor phosphorylation and desensitization as well as study the molecular structure of mu opioid receptor. We employed molecular biology and cell biology techniques combined with pharmacological methods to accomplish these research goals in three aspects. In the first part, we characterized agonist-induced mu opioid receptor phosphorylation events in brain tissues. We found that in the thalamus of morphine-tolerant rats, both the basal and agonist stimulated receptor phosphorylation levels were enhanced compared with those observed in normal rats. The enhanced phosphorylation level correlated well with the desensitization of their cellular response. These findings demonstrate that agonist induced mu opioid receptor phosphorylation Plays an important role in receptor desensitization, which may contribute to the mechanisms underlying tolerance and dependence. In the second part, we determined the role of the C-terminus in mu opioid receptor phosphorylation and desensitization by C-terminal deletion and point mutation of the receptor. Our data suggest that the C-terminus is an important domain involved in the 4 opioid receptor phosphorylation and desensitization. We further found that threonine 394 is a crucial residue that is required for agonist-induced mu opioid receptor phosphorylation and desensitization. In the last part, we investigated the effects of sulfhydryl reagents on mu receptor binding and identified that the cysteine residues in TMIII (C161), TMIV (C192), TMV (C237) and TMVII (C332) of the receptor that are critical cysteines responsible for the sensitivity of mu opioid receptor binding to sulfhydryl specific reagents. These four cysteines may be located in or near the receptor binding site. These findings provide important information for understanding the molecular mechanisms of interaction between the mu opioid receptor and its ligands.
    • The influence of aging on brain muscarinic receptors in the rat

      Surichamorn, Wanida; El-Fakahany, Esam E. (1991)
      Cognitive dysfunction is a dominant symptom that is generally reported in elderly as well as in Alzheimer's disease patients. The decline of such function is not recognized only in human beings but also in non-human mammalian species such as mice, rats, and monkeys. To explain this abnormality, several neurotransmitter systems have been demonstrated to decline accompanying the aging process and also they are postulated to play an important roles in the memory process, such as cholinergic, dopaminergic and noradrenergic system. However, a large number lines of evidence supported that the central cholinergic system especially in basal forebrain plays a pivotal role in memory function. These studies were designed to answer questions related to the effects of aging on the muscarinic receptor levels as well as its responsiveness upon the receptor stimulation. Fisher 344 rats were utilized as the study model since a large number of behavioral lines of evidence reported that the aged animals show a memory impairment similar to those in old humans. In this study, it is clear that there are age-associated specific alterations in the density of cell surface muscarinic receptors rather than the total receptors without changing their affinities in certain brain areas such as striatum and cerebral cortex. However, the alterations in receptor number is not associated with changes in the proportion as well as the affinities of pirenzepine-high affinity (M{dollar}\sb1{dollar}) and -low affinity (M{dollar}\sb2{dollar}: old nomenclature) in striatum, hippocampus and cerebral cortex with advancing age. In addition, the allosteric sites which are modulated by gallamine are not disturbed during the aging process. Furthermore, age-related changes in two main second messenger systems of muscarinic receptors such as the inhibition of adenylate cyclase (which may be linked to acetylcholine release) and PI hydrolysis (thought to play a pivotal role in the memory process) were studied. There were no age-related changes in the inhibition of Forskolin-stimulated cyclic AMP formation which contrasted to the well documented decline in acetylcholine release during aging, suggesting that these two responses are not linked to each other. Moreover, the data from the studies indicated that PI hydrolysis mediated by brain muscarinic receptors is not sensitive to age-induced changes in brain function. In addition, such receptor function was equally sensitive to blockade by phorbol esters and tetrodotoxin in young and aged rats. However, subtle changes might occur in discrete brain areas or in individual inositol phosphate species, especially inositol 1,4,5-trisphosphate, which cannot be detected by the methods employed in the present studies. It is also interesting to note that there are no age-related changes in the muscarinic receptor plasticity upon agonist pre-exposure, suggesting that treatment of memory deficit in the elderly with cholinermimetics should be performed with caution.
    • Interactive effects of MK-801 and morphine on EEG, EEG power spectra and behavior in rats: Acute, tolerance and dependence studies

      Haberny, Kathleen Anne Y.; Young, Gerald A. (1993)
      Recent studies investigated an interaction between MK-801 and morphine-induced analgesia which originates in spinal and supraspinal subcortical areas of the CNS, particularly in the periaqueductal gray. Interactive effects between MK-801 and morphine-induced cortical EEG that coincides with behavior generated in mesolimbic areas have not been demonstrated. The present study first investigated an acute interaction between MK-801 and morphine-induced effects on cortical EEG and EEG spectral parameters in rat. Rats were administered one of five doses of MK-801 (0.003-0.3 mg/kg, i.p.) prior to morphine (3 mg/kg, i.v.), or MK-801 (0.1 mg/kg, i.p.) prior to an increasing cumulative morphine dose over a period of 120 minutes (5-120 mg/kg, i.v.). MK-801 pretreatment produced a significant dose-response effect and increased morphine-induced global spectral power (mV{dollar}\sp2{dollar}/Hz), duration of morphine-induced high voltage slow wave EEG bursts and latency to sleep onset. MK-801 significantly decreased morphine-induced mean frequency, mobility, complexity and edge frequency, and shifted the relative distribution of total power as a function of frequency band to lower frequencies, increasing power in the 0-5 Hz range. Significant interaction effects were found for all spectral parameters except peak frequency. Pretreatment with MK-801 shifted the cumulative morphine dose-response curve for total power to the left and for complexity to the right. The results demonstrated that MK-801 potentiates morphine-induced effects on EEG and EEG spectral parameters quantitatively in rat brain and produces qualitative changes in the EEG response to morphine. A potential interaction between MK-801 and morphine tolerance and dependence was further investigated. Rats were treated for seven days with morphine alone or with morphine and MK-801. Control groups received chronic MK-801 alone or saline. On day eight all rats received morphine alone followed by naloxone. An additional group received chronic morphine alone, followed by MK-801 on day eight prior to morphine and naloxone. Co-treatment significantly accelerated the development of tolerance to morphine-induced total power and latency to sleep onset. MK-801 co-treatment decreased, where chronic morphine alone increased, the excitatory response to morphine. Chronic co-administration had no effect on the EEG and behavioral response to naloxone precipitated morphine withdrawal, but acute MK-801 in morphine tolerant animals prevented the behavioral signs of morphine withdrawal. The results support recent reports that chronic morphine treatment produces supersensitivity to glutamate in the rat cortex and alteration in mesolimbic dopamine levels which are modulated by glutaminergic activity. In conclusion, acute co-treatment with MK-801 significantly potentiates morphine-induced EEG, EEG spectral parameters and behavior, and attenuates the behavioral signs of withdrawal from morphine following chronic morphine administration. Chronic co-treatment, furthermore, accelerates the development of tolerance to morphine-induced effects on these parameters.
    • Pharmacological treatment during status epilepticus: Metabolic and electrographic studies

      Jones, Bruce Edward; Buterbaugh, Gary G. (1992)
      The present study examined the effect of diazepam, pentobarbital, THIP (4,5,6,7-tetrahydroisoxazolo-(4,5c)-pyridone-3-d)), valproic acid and MK-801 treatment on metabolic, electrographic and behavioral components of a pilocarpine facilitated model of status epilepticus (pfSE). This study demonstrated that amygdala kindled rats pretreated with a non-convulsive dose of pilocarpine and electrically stimulated produce a stable seizure characterized by high-amplitude, fast-frequency EEG and increased ({dollar}\sp{lcub}14{rcub}{dollar}C) -2-deoxy-D-glucose ( ({dollar}\sp{lcub}14{rcub}{dollar}C) -2DG) uptake. Additionally, the present study demonstrated diazepam treatment during pfSE resulted in a dose-dependent decreased ({dollar}\sp{lcub}14{rcub}{dollar}C) -2DG uptake. The somatosensory cortex appeared most sensitive whereas the amygdala, entorhinal cortex, reuniens nucleus, lateral septum and claustrum-endopiriform exhibited a resistance to diazepam with respect to ({dollar}\sp{lcub}14{rcub}{dollar}C) -2DG uptake. The effect of diazepam was attenuated by the selective benzodiazepine antagonist Ro-15-1788. As with diazepam pentobarbital reduced regional ({dollar}\sp{lcub}14{rcub}{dollar}C) -2DG uptake, however, the effect was not dose-dependent. THIP, valproic acid, and MK-801 did not alter the regional ({dollar}\sp{lcub}14{rcub}{dollar}C) -2DG uptake pattern during pfSE. Except for pentobarbital no compound administered resulted in alterations in EEG. Therefore diazepam treatment was the only treatment which resulted in reductions in ({dollar}\sp{lcub}14{rcub}{dollar}C) -2DG uptake without attenuation of EEG. Furthermore the data did not establish a link between potentiation of GABA receptor-mediated inhibition of neuronal activity and the effect of diazepam. However, there was evidence to suggest that kindling is important to the effect. This study also provides evidence that increased ({dollar}\sp{lcub}14{rcub}{dollar}C) -2DG uptake was associated with high dose pilocarpine-induced SE. However, these data did not indicate that diazepam treatment resulted in reduced ({dollar}\sp{lcub}14{rcub}{dollar}C) -2DG uptake in this particular model. In summary these data clearly demonstrate a dissociation of ({dollar}\sp{lcub}14{rcub}{dollar}C) -2DG uptake and electrical activity as a result of diazepam treatment. Furthermore these data suggest that diazepam mediates this effect through a specific receptor-mediated effect, however, no connection was established between the effect of diazepam and potentiation of GABA receptor-mediated inhibition of neuronal activity but it does appear as if kindling is necessary.