Floyd Sallee - US grants

This candidate wishes to systematically expand his investigative capabilities in basic and clinical psychopharmacology to become a pediatric psychopharmacologist and to explore innovative treatment strategies in content areas of Attention Deficit Disorder and Tourette's Syndrome. These content areas were chosen to accomplish two long term goals of the candidate: (1) the characterization of the drug level vs. clinical response relationship in children and (2) in vivo exploration of mechanism of drug action in children via neuroendocrine and behavioral assessment measures. Drugs of interest include pemoline, which will be studied in vivo and in vitro in a rat model as well as in hyperactive children, and pimozide and haloperidol which will be studied in children with Tourette's Syndrome. Preliminary data obtained by the candidate showing an inverse, in-phase relationship between pemoline and prolactin concentrations in hyperactive children needs further characterization both in clinical studies and in an animal model. The clinical significance of such a relationship is that it defines the time course of pemoline's effect as an indirect dopamine agonist and may correlate with clinical response to pemoline. Work to be completed over the first three project years include (1) Define the pharmacokinetic profile of pemoline in the rat, (2) Study the pharmacologic effect of reserpine and a methyl-p-tyrosine pretreatment on pemoline's action as defined by motor activity and prolactin secretion in the rat, (3) Examine the binding of high specific activity 3H pemoline in rat brain, (4) Establish whether pemoline inhibits its own metabolism in hyperactive children, (5) Characterize the relationship of pemoline level to prolactin response after both acute and three weeks of pemoline, (6) Develop a sensitive and specific RIA assay for pimozide that will accurately measure from 0.5-50 ng/ml, (7) Characterize the pharmacokinetics of pimozide in children with Tourette's. Work to be completed in years 4 and 5 of the award include (1) Characterization of the relationship between steady-state pemoline concentration and clinical response in hyperactive children in a double-blind, placebo controlled, cross-over study, and (2) Comparison of therapeutic efficacy of haloperidol and pimozide for the control of Tourette's and an estimate of the therapeutic ranges for these drugs in the pediatric age group.

Tourette Syndrome (TS) is a debilitating and chronic neuropsychiatric disorder. Haloperidol is currently the drug of first choice in the treatment of TS but it frequently causes severe side effects which hamper its therapeutic usefulness. Pimozide has only recently been approved by the F. D. A. for use in TS and it is currently used only if treatment with haloperidol is unsuccessful. Pimozide has been demonstrated to be efficacious in TS but a direct comparison with haloperidol has not been done. The literature suggests that pimozide may have fewer side effects and may produce less cognitive impairment associated with therapeutic doses than haloperidol. The specific aim of this proposal is to determine the relative efficacy of pimozide and haloperidol utilizing a placebo controlled crossover design in a group of thirty six children suffering from TS. Three treatment conditions (haloperidol, pimozide, placebo) will be randomly assigned in a balanced fashion with two week washout periods between crossovers and six weeks on each treatment. The protocol will mimic clinical practice by aiming for a 70% reduction of tic symptoms using a flexible dose paradigm below 12 mg/day for each drug. The individualized dosing will allow relative toxicity to be compared at equivalent efficacies. A comparison of pimozide and haloperidol at their "most efficacious" dose will be made from data gathered at the end of six weeks on each medication. Characteristics unique to this proposal in comparison to the literature are the following: 1) a multidimensional approach to assessment of differential effects of haloperidol and pimozide via neurologic, cognitive, social, behavioral, and school performance outcome variables, 2) dose-response assessment to provide therapeutic and toxic ranges for these drugs in TS and where appropriate, therapeutic plasma concentration ranges to facilitate a targeting of the dose into a therapeutic range to avoid side effects. The proposal has important clinical implications in the treatment of children with TS if pimozide proves to have significant advantages over haloperidol in terms of efficacy and fewer side effects.

Identification and characterization of the cocaine receptor in the CNS is a necessary step toward the development of efficacious treatment agents for cocaine abuse. Preliminary evidence suggests that the high affinity [3H] cocaine binding site in brain is associated with the dopamine (DA) transporter. The reinforcing properties of cocaine and cocaine analogs correlates with their potency of DA transport inhibition and affinity for the DA transporter site, implicating this site as the "cocaine receptor". The specific identification and biochemical characterization of this receptor has not yet been achieved. One of the receptor site via photoactive radiolabeled ligands which upon photoincorporation would be covalently attached to the receptor. The specific aim of this proposal is the development and pharmacologic testing of a number of radiolabeled photoaffinity ligands for the cocaine receptor. We have selected the aryldialkylpiperazine "GBR12909" as a model compound from which to synthesize such a photoaffinity label because of its high affinity for the [3H] cocaine binding site and the DA transporter. Our laboratory has generated a limited number of pilot aryl azide GBR compounds which suggest that the production of a photoaffinity ligand for the cocaine receptor is feasible. We propose to generate and select GBR photoaffinity ligands with high affinity for the cocaine receptor which will be subsequently radiolabeled with 125I. We will utilize the [125I]GBR-N3 compound to identify the cocaine receptor by chromatographic and electrophoretic techniques. Selectivity and specificity of photoincorporation of the [125I]GBR-N3 will be evaluated as well as preliminary pharmacology and biochemistry of the labeled site to demonstrate it to be the cocaine receptor.

Identification and characterization of the dopamine (DA) transporter containing the cocaine binding site (cocaine receptor) in the CNS is a necessary step toward the development of efficacious treatment agents for cocaine abuse. This proposal will isolate and purify a putative DA transporter protein for protein sequencing using a GBR affinity chromatography method, and will identify its cDNA. The investigators have purified a glycoprotein of Mr 62 kDA which retains a pharmacologic profile suggestive of the DA transporter. Purification in sufficient yield for protein sequencing is now feasible. Western blots and immunoprecipitation studies of the polyclonal rabbit antibody against the protein suggests recognition of an epitope at a similar molecular weight. Screening of an expression library in phage lambda-gt11 from human substantia nigra using the polyclonal antibody has demonstrated 8 positive clones from an initial screen of 200,000 plaques. Rescreening of the library with the putative clones is presently in progress. Northern blots indicate positive signals localized to the substantia nigra using these clones. RACE PCR is now underway to obtain full length cDNA sequence utilizing primers from two sources 1) partially sequenced clones; 2) partial peptide sequence from the GBR-affinity purified protein. Specific aims to be accomplished in this proposal are: 1a) complete pharmacologic characterization of the affinity purified DA transporter and reconstitution functional studies of the protein, 1b) protein sequencing; 2) full characterization of the rabbit polyclonal antibody including immunohistochemistry, 3) expression cloning of the DA transporter with subcloning and sequencing using primer extension to characterize inserts of subclones presently identified. Partial inserts will be used to rescreen the library to obtain full length clones. Full length cDNA specificity, which may be available from the initial subclones, will be confirmed using Northern hybridization and rescreening of the original expression library. RACE PCR will be used to generate 3'and 5' ends from substantia nigra RNA template. Expression of full coding sequence for DA transporter in a eukaryotic system such as COS cells which do not normally express cocaine binding, followed by pharmacologic and functional assessment of high affinity cocaine binding and dopamine uptake capability in the transformed COS cells. Expression in a Baculovirus system is planned to provide large quantities of transporter protein for protein sequencing and incorporation into liposomes for functional studies. Long range goals will include site directed mutagenesis and a survey of regional expression and ontogeny of brain mRNA levels coding for the cocaine receptor.

human therapy evaluation; mental disorder chemotherapy; anxiety disorders; fenfluramine; middle childhood (6-11); yohimbine; adolescence (12-20); clonidine; psychobiology; somatotropin; neurobiology; cortisol; neuroendocrine system; prolactin; adrenocorticotropic hormone; neuropharmacology; clinical research; human subject; intravenous administration;

To test the neuroendocrine response in 48 obsessive-compulsive patients aged 16-45 to low dose (12.5 mg) parenteral CMI. Also to determine the acute efficacy of high dose (200mg) bolus CMI vs. placebo saline to relieve OCD.

To test the neuroendocrine response to low-dose (12.5 mg) parenteral CMI in disorders where serotonin has been implicated (e.g., Tourette's disorder, anxiety disorders, major depression) to determine if it can discriminate patients from controls.

Dopamine (DA) blocking neuroleptics are now standard pharmacotherapy of Tourette disorder (GTS) in children. This approach is based on a hypothesis of DA hyperfunctioning in GTS. This proposal seeked to enhance the treatment of GTS by evaluating a DA agonist strategy with pergolide which may be superior to dopamine blockade. If this strategy has merit, it would: 1) redefine our treatment approach to GTS and 2) enhance the risk/benefit ratio for children by reducing exposure to neuroleptics. This particular proposal will facilitate the development of pergolide mesylate in a new indication in GTS by establishing its safety and efficacy in this population. At present, pergolide's safety and efficacy is unknown in children and its sole indication is in Parkinson's disease.

Attention Deficit, Hyperactivity disorder (ADHD) is a common mental health disorder of significant morbidity for which approximately 100,000 children yearly are treated with clonidine (CLON) in the US, either as an alternative to psychostimulants, or as combination therapy. Adequate ADHD efficacy and safety data are lacking and CLON is not FDA approved for ADHD. Rationales for combination with methylphenidate (MPH) are that it may be synergistic, may extend the stimulant effect, and/or that CLON may address certain core symptoms not treated by stimulants alone. No data support these rationales, yet the combination is increasingly being prescribed despite reports of serious adverse events linked to combination therapy. CLON has yet to be comprehensively studied in primary ADHD, but an NINDS sponsored evaluation of the safety and efficacy of CLON alone and in combination with MPH is currently being conducted in children with Tourette Syndrome with comorbid ADHD (TACT). TACT subjects however may be predominantly "inattentive" subtype, in contrast to combined type, most commonly seen in primary ADHD. TACT data also cannot be extrapolated to primary ADHD because treatment with CLON in TACT is dually targeted to the treatment of both tics and ADHD such that dosing interval and potentially safety findings (if adverse events are dose related) may not be readily applied to primary ADHD. Efficacy and safety data from TACT therefore may be inadequate for primary ADHD treatment, for which the majority of CLON is prescribed. The clinical trial machinery and expertise of the TACT team however is uniquely positioned to study CLON in primary ADHD. Advantages would be: 1) reduced cost by using TACT existing infrastructure; 2) an opportunity to pool safety data across studies such that low frequency adverse events may be detected; 3) to compare therapeutic outcomes in primary ADHD to ADHD with comorbid Tourette Syndrome to determine if CLON has differential effects in "inattentive subtype" versus "hyperactive/impulsive" or combined subtypes; and 4) to determine if chronic treatment with MPH can contribute to the development of tic disorder. We propose a multicenter, controlled study of CLON, MPH, and CLON plus MPH for children with primary ADHD. The study will A) determine the safety and efficacy of CLON for the treatment of ADHD in children; B) evaluate the safety and efficacy of combined CLON+MPH for treatment of ADHD compared to standard MPH therapy; C) provide a lyr extension for all subjects to follow-up tic development during course of the study.

DESCRIPTION (provided by applicant): Our network group seeks to formalize a collaboration of neuroscientists, geneticist, pharmacologists, pediatricians, and child psychiatrist so as to transcend the limitations of our present conceptualization of ADHD etiology by concentrating on non-clinical phenotypes. Non-classical phenotypes were chosen based on their shared pathophysiology involving circuitry which is amenable to "circuit-testing" of behavior and pharmacologic dissection in animal models as well as the clinic. These ADHD phenotypes involve dopaminergic (DA) pathophysiology as a core feature, and share common cortical-limbic glutamatergic neuronal (CGN) circuitry in symptom generation. Each phenotype has heuritic value to bring an understanding not only to ADHD "outliers" but for classical ADHD through the identification of shared mechanisms. One such shared mechanism we hypothesize is the glutamatergic valence of key cortical-limbic pathways (Carlsson 2000). The lead-exposed ADHD phenotype we propose as a prototype for determining the interaction of DA genotype (DRD4, DRD5, DAT) and environment producing ADHD symptoms and impairment. In our network, we have developed interposing projects at the behavioral/phenomenologic and animal model-genetic determinant levels to inform a translational "proof of concept" clinical trial. This feasibility trial will estimate effect sizes for the cortical-limbic noradrenergic (NE) releasing drug, atomoxetine. Pharmacologic dissection of ADHD phenotypes is now possible using direct DA agonists/antagonists as well as indirect agents impacting cortical-limbic glutamatergic neuronal (CGN) circuitry like atomoxetine. Major strengths of this proposal include: 1) Strong, ongoing collaborative relationships between network members; 2) novel and testable hypothesis regarding non-classical ADHD phenotypes encountered in clinical practice from which "proof of concept" pharmacologic trials can be initiated; 3) an animal model that is well characterized and highly exploitable for "circuit-testing" of antipodal behavioral features of the ADHD-TS phenotype; 4) a unique opportunity to study the neurobehavioral outcomes of an ongoing lead-exposed cohort of 212 children as the cohort reaches the time (school-age) when a clinical diagnosis of ADHD typically occurs; 5) examination of gene-environment interactions with DA-associated polymorphisms linked to ADHD phenotype expression, and 6) strong, extra-network linkages with the pediatric pharmacologic research units (PPRU) infrastructure, an animal model behavioral phenotyping laboratory, and a Howard Hughes Bioinformatics Center to expand on Arrant findings and "proof of concept' trials.

DESCRIPTION (provided by applicant): Attention-Deficit Hyperactivity Disorder (ADHD) is a behaviorally defined phenotype with patterns of deficits involving attention and inhibition characterized by variability in presentation, pervasiveness, and impairment. Pathophysiologic models of ADHD have advanced through intermediate constructs termed endophenotypes which are quantifiable, grounded in an understanding of the relevant neuro-circuitry, and simplified to be more closely linked to gene expression. We hypothesize that abnormal intracortical inhibition (ICI) functions as a physiologic transducer, a mediating or moderating element underlying many endophenotype constructs in ADHD. A biomarker of GABAergic, dopamine-sensitive, short-interval intracortical inhibition (SICI), is available through emerging technology involving paired-pulse, transcranial magnetic stimulation (pTMS) of motor cortex. Our group has studied SICI in children and adolescents with ADHD with regard to safety, stability, and test-retest reliability. Evidence reveals cortical inhibition (SICI) to be inversely correlated with ADHD symptom severity. As SICI is abnormal in ADHD, our studies suggest SICI change after ATX may signal a physiologic compensation which may mediate subsequent clinical response. We propose these specific aims: 1) To evaluate Intracortical Inhibition (SICI) measured by pTMS, as a marker of the hyperactive-impulsive dimension and symptom severity; 2) To determine cognitive correlates of SICI change relevant to a well characterized ADHD endophenotype of response suppression/inhibition by time-locking elements of a Stop Task to pTMS; 3) To characterize the effects of four weeks of atomoxetine (ATX) treatment on ADHD and cortical inhibition. We will assess 120 7-12 year old children with ADHD in a double-blinded, placebo controlled design (ATX vs PLB) evaluating expanded neurophysiological inhibitory markers of transcallosal cortical inhibition (e.g. ISP) along with SICI (aim 3a), and, to follow up on our prior findings related to dopamine transporter polymorphisms, we will assess the contributions of multiple possibly ADHD-related genes (aim 3b). This aim will evaluate whether SICI change following 4 weeks of treatment with ATX predicts treatment response (change from baseline ADHD Rating Scale). Controlling for mental state in this proposal by using the Stop Task may elucidate our seeming paradoxical finding of decreased SICI with ATX response (Gilbert 2007). This aim, though exploratory, has the potential for high clinical impact, determining whether genomic information can be combined with pTMS to more accurately predict clinical response to ATX, as treatment now is delayed, and unpredictable without phenotypic predictors of response. PUBLIC HEALTH RELEVANCE: Attention-deficit, hyperactivity disorder is the most common behavioral disorder of childhood produces significant morbidity in academic, vocational and psychosocial outcomes. ADHD is complex in etiology, with many neurobiologic substrates, one of which is short-interval intracortical inhibition (SICI). SICI measured by transcranial magnetic stimulation (pTMS) of motor cortex has been shown to be inversely correlated with ADHD symptom severity. This proposal will further characterize both the physiologic relevance of SICI for ADHD as well as its utility to predict response to ADHD treatments such as atomoxetine.