Analytical Psychopharmacology

Thomas B. Cooper, M.A., Chief of Psychiatric Research
Amiram I. Barkai, Ph.D., Research Scientist VI
Basalingappa L. Hungund, Ph.D., Research Scientist V
Ee-Sing Lo, Ph.D., Research Scientist V
Hana Novacenko, M.S., Research Scientist III
Raymond F. Suckow, Ph.D., Research Scientist V

Marc Laruelle, M.D., Research Psychiatrist
Henry Huang, Ph.D., Research Scientist IV
Dah-Ren Hwang, Ph.D., Research Scientist IV
Diana Martinez, M.D., Research Psychiatrist
Osama Mawlawi, Ph.D., Research Scientist III
Mark Slifstein, Ph.D., Mathematician
Rikki Waterhouse, Ph.D., Radiochemist
Eric Zarahn, Ph.D., Statistician
Yolanda Zea-Ponce, Ph.D., Radiochemist

The department continues to serve as a core laboratory facility in two NIMH and one NIDA Clinical Research Centers, a NIDA Medication Development Research Unit, an NIMH Research Unit for Pediatric Psychopharmacology, and several large multicenter clinical studies. In addition, the department has collaborated with many academic research centers within and outside of the USA. An extension and increase in scope of these collaborative efforts has been brought about by the transfer of Dr. Marc Laruelle and his imaging group to this department. This group will form the Functional Brain Mapping Division (FBMD) within Analytical Psychopharmacology (APL). The APL has worked with Dr.Laruelle on many protocols in the past and we welcome our new departmental colleagues and anticipate even more exciting and productive collaborations in the future.

The department and new division have conducted clinical and basic research in an ever widening variety of psychiatric disorders, e.g., anxiety, depression, obsessive compulsive disorder, schizophrenia, Alzheimer's disease, and substance abuse in all age groups. We continue our long established assay development program to explore the pharmacokinetics and pharmacodynamics of new psychotrophic drugs and their metabolites and, where appropriate, their enantiomers. Basic work involves investigations at the cellular level of the interaction of alcohol with the cannabinoid (CB1) receptor. Recent work in each of these areas is briefly reviewed. The FBMD program is described separately.

Dr. Suckow and his staff have developed many methods for the HPLC assay of psychotrophic drugs. Analyses of plasma amino acids are continuing to generate useful data in the management of certain psychiatric disorders. To determine the role of these substances in specific disorders, an increasing number of research protocols require the quantitation of either the large neutral amino acids group (valine, leucine, etc.), or the excitatory amino acids (glutamate, glycine). A procedure for the quantitation of carnosine, homocarnosine and N-acetylhistidine has been developed as part of a collaborative effort in a pilot study to identify the possible functions of the intercellular transport of these substances, as well as that of NAA and NAAG. NAA, as well as other N-acetylated amino acids, is implicated in a number of pathophysiological conditions in brain.

Preliminary work has begun in the development and validation of a new analytical procedure for the analysis of plasma kavalactones, the pharmacologically active constituents of Piper methysticum, or Kava. Kava, a popular natural herbal remedy, is well known for its anti-anxiety, analgesic, anesthetic, sedative, muscle-relaxant, and anti-convulsive properties. This is part of a pilot study at CPMC-Neurological Institute to determine the pharmacokinetics (PK) and bioavailability of these substances in plasma as it relates to dose and efficacy in humans. To date, there has been no published PK data for Kava in humans.

Extensive work in the pharmacokinetics and metabolism of bupivacaine in a variety of tissues has been on-going as part of a NIDA funded study (Dr. Morishima, Principal Investigator) which is intended to address the possible interaction between cocaine and metabolites and bupivacaine and metabolites. New data regarding the distribution and disposition of bupivacaine metabolites (3-hydroxybupivacaine, 4-hydroxybupivacaine, and PPX) have been generated. Other protocols requiring the plasma determinations of fluoxetine and its metabolite, sertraline and its metabolite, paroxetine, risperidone and the active 9-hydroxy metabolite, lorazepam, modafinil, olanzapine, fluvoxamine, ketamine and metabolites, and bupropion and metabolites are continuing.

Dr. Lo continues his extensive collaborations within the institute and, with the neuroendocrine section, has been involved in the assay of several thousand samples for a variety of neurotransmitters, neurotransmitter metabolites, melatonin (saliva and plasma), dexamethasone, betamethasone, cortisol (saliva, plasma, and urine) prolactin, growth hormone, ACTH (human and rat), TSH, FSH, LH, estradiol and corticosterone in plasma and/or urine from clinical and animal studies.

The assay of transthyretin (Dr. J. Gorman) has been validated and a large number of spinal fluid samples analyzed in the past year. Current developmental work includes the analysis of spinal fluid CRH and validating methodology for the assay of Substance P (Drs. Mann and Stanley), c-AMP and KREB in fibroblasts obtained via cell culture of skin biopsies from normal volunteers and depressed patients (Dr. Sackeim).

We have spent considerable effort to standardize and validate a method for salivary melatonin. This methodology has proven to be a major breakthrough in the ongoing studies of Seasonal Affective Disorder (SAD), where light therapy has been shown to reverse phase shifts in melatonin secretion (Dr. Terman), Circadian rhythm disruption via jet-lag (Dr. Boulos), and hemodialysis phototherapy with Dr. Dan Oren of Yale University. Until now these experiments required that a patient stay overnight at the Institute with multiple blood samples collected throughout the evening and overnight which is time consuming, labor intensive, costly, and therefore self-limiting. The use of saliva sampling has allowed the patient to collect multiple samples at home or on a plane thus increasing the number of patients who will volunteer for such a study and reducing operational costs.

We continue the assay of plasma catecholamines using a triple cell amperometric detector HPLC-validated system developed in our lab several years ago. In 2000, collaborative studies included protocols with Dr. Pelton, Dr. Marshall, Dr. Sloane, and Dr. Shapiro, and the pharmaceutical companies Wyeth-Ayerst and Alkermes.

Drs. Hungund and Basaravajappa have continued their investigation of the mechanisms involved in development of tolerance to and dependence on ethanol (EtOH). They have now identified yet another endocannabinoid, namely, 2-arachidonyl glycerol (2-AG) in primary neuronal cultures chronically exposed to EtOH. This is in addition to this group's previous findings of anandamide accumulation in the brain membranes of mice similarly treated with EtOH. They have also demonstrated that differences exist in the distribution of CB1(cannabinoid) receptors in EtOH-preferring (C57/BL) and EtOH-avoiding (DB1) strains of mice. This finding supports the hypothesis that CB1-ergic mechanisms may be involved in EtOH drinking behavior. They are also testing this hypothesis further by utilizing pharmacological tools such as CB1 receptor agonists and antagonists against the development of tolerance and abusive drinking behaviors. Preliminary studies appear to indicate that the animals preferred alcohol when they were exposed to a CB1 receptor agonist and the administration of a CB1 receptor antagonist reduced alcohol intake. This is the first evidence reported which strongly supports the participation of cannabinoidergic mechanisms in alcohol addiction. Studies are underway to establish a definitive link between the cannabinoidergic system and EtOH's pharmacological and behavioral effects through the use of a mouse strain lacking the CB1 receptor gene (CB1 knock-out mice).

In a collaborative effort with Dr. Arango's group of the Neuroscience division, the cannabinoid receptors in the brains of EtOH-tolerant mice have now been mapped using autoradiographic procedures. Preliminary studies seem to suggest that striatum and cerebellum are the regions most affected (receptor down-regulation) in EtOH-tolerant mouse brain. Efforts are underway to test if CB1-ergic mechanisms are involved in depressive illness since co-morbidity between alcoholism and depression is widely recognized. Similar analyses are to be performed on postmortem human brain tissue that Drs. Mann and Arango have collected for other studies.

Dr. Barkai continues work in collaboration with Dr. A. Dwork and Dr. Hungund on his protocol "Brain phospholipids and mental illness: Dynamic aspects". The study involves the estimation of the turnover of polyunsaturated fatty acids in membrane phospholipids in specific brain areas of deceased patients with schizophrenia. Dr. Dwork, a neuropathologist at PI, provides frozen tissue samples after autopsy and Dr. Hungund of this department collaborates in the lipid analyses. These experiments examine rates of deacylation and reacylation of membrane phosholipids and age or time from death to autopsy. These data in turn are to be used to test the hypothesis that alterations in turnover rates of membrane phosholipid produce molecular lesions in neuronal membranes positively correlated with age and disease. These studies are expected to enhance our understanding of the molecular mechanisms involved in schizophrenia.

During the past year the division has continued to focus on 1) developing several brain imaging methods aimed at studying the living human brain and 2) applying these techniques to study alterations in brain function associated with major mental illnesses. Imaging modalities involved PET neuroreceptor imaging, magnetic resonance spectroscopy (MRS), and functional MRI (fMRI). Clinical studies were performed in collaboration with several clinical programs from within the Department of Psychiatry, such as programs in schizophrenia, anxiety disorders, and substance abuse, as well as with clinical programs from other institutions (Mt Sinai, Yale).

Research Programs and Research Projects The neuroreceptor Positron Emission Tomography (PET) program, initiated in 1997, fully matured in 2000. The key event of the year 2000 was the completion of the construction of a new radiochemistry laboratory in the basement of Milstein, next to the cyclotron. This laboratory became functional in the summer of 2000. Until then, the radiochemistry work was performed in a laboratory shared with a private company, PET-NET, and we had access to only one hot cell. The new laboratory includes three hot cells, and is dedicated to research. Thus, we experienced this year a remarkable increase in our ability to develop and produce radiotracers for PET imaging. We are extremely grateful to the Lieber family, who provided an important financial contribution to this major investment. With this new laboratory, we are now in position to take full advantage of PET technology to explore chemical imbalances associated with major psychiatric conditions.

The other major event of the year for our division was the organization of the Neuroreceptor Mapping 2000 Conference here at Columbia. This conference takes place every other year, and gathers scientists from all over the world involved in the development and implementation of neuroreceptor imaging with PET. Two years ago, our site was selected to host the 2000 edition of the meeting, which was largely organized by Drs. Abi-Dargham and Mawlawi. The conference was a great success, with exceptional representation of the international PET community leading to scientific communication and exchanges of the highest quality.

The paper from Abi-Dargham, et al., published in the PNASU and reporting the results of our investigation of synaptic dopamine level in patients with schizophrenia, was selected for the cover and subject to editorial comments in PNASU and Lancet. Dr. Kegeles paper reporting modulation by ketamine of amphetamine-induced dopamine release in humans was published as a priority communication in Biological Psychiatry.

A total of 230 PET neuroreceptor-based studies were performed in 2000, including 53 in nonhuman primates and 177 in humans. Studies in baboons were performed to evaluate several new and original radiotracers designed by the radiochemistry team, including [¹¹C]MHA, a new 5-HT_1A agonist, [¹¹C]DAPP, a new 5-HT transporter ligand, [¹¹C]GLY4, a new glycine site antagonist, [¹¹C]BP897, a new dopamine D3 receptor antagonist and [¹¹C]PMP, a new dopamine D4 receptor antagonist. In addition, studies in baboons were conducted to prepare the implementation in humans of tracers previously used in other PET centers, but currently unavailable here, such as the 5HT2A antagonist [¹¹C]MDL100907 and the dopamine transporter blocker [¹¹C]cocaine. Baboon studies were also designed to evaluate potential new pharmacological interventions. For example, we evaluated the effect of the glutamate metabotropic receptor agonist LY354740, a drug developed for the treatment of anxiety and negative symptoms of schizophrenia, on dopamine transmission using PET. In 2000, two new ligands became available for human use. [¹¹C]MDL100907 was evaluated in healthy volunteers and showed excellent properties as a radiotracer to quantify 5HT2A receptors. We also performed the first human scans at Columbia with the dopamine transporter SPECT ligand [¹²³I]ß -CIT.

Because of the availability of the new high resolution PET camera installed in 1999, we could initiate studies aiming at differentiating dopamine transmission in the dorsal regions of the striatum (where dopamine is mostly involved in control of movement) versus the ventral regions of the striatum (where dopamine mediates pleasure, motivation, and reward). The development of new image analysis methods involving image fusion and correction for partial voluming effects were essential to reach this high level of resolution. Studies conducted in healthy volunteers by Drs. Mawlawi and Martinez demonstrated the reliability of this measurement. These studies demonstrated that dopamine transmission in the ventral regions of the striatum is selectively activated by amphetamine, and that this activation mediates the euphoria elicited by the drug.

In schizophrenia, studies led by Dr. Abi-Dargham characterized dopamine D1 receptor and distribution in the dorso-lateral-prefrontal cortex (DLPFC), and how these alterations relate to cognitive deficits experienced by these patients. These studies are conducted in collaboration with the Schizophrenia Research Unit and the MHCRC Schizophrenia Research Center. Initial results demonstrate a selective increase in the density of D₁ receptor in the DLPFC in patients with schizophrenia, and that this upregulation is extremely predictive of poor performance on tests involving working memory. If replicated, this finding could help to identify patients most likely to benefit from D₁ agonist therapy.

In cocaine abuse, we are collaborating with Drs. Kleber and Fischman's group in a study aimed at measuring dopamine transmission in the ventral striatum and at characterizing the relationship between limbic dopamine systems and cocaine self-administration. So far, these studies conducted by Dr. Martinez reveal a selective and almost complete blunting of mesolimbic dopamine transmission in chronic cocaine abusers. This observation might reflect long-term damage of the mesolimbic DA system associated with years of chronic cocaine exposure, and might explain why cocaine abuse leads to decrease potency of natural rewards such as food and sex.In anxiety disorders, we conducted studies of serotonin and dopamine transmission in patients with social phobia and obsessive compulsive disorders in collaboration with Drs. Liebowitz and Gorman. One of the key findings of this year was that therapeutic dose of the selective serotonin uptake inhibitor paroxetine was associated with complete occupancy of the serotonin transporter. This study, conducted by Drs. Kent and Coplan, is the first report of serotonin transporter occupancy level achieved during treatment with an SSRI, and has several implications for treatment of depression and anxiety disorders.

Magnetic Resonance Spectroscopy (MRS) Program In collaboration with Drs. Shungu and Hetterington, Dr. Kegeles continued to characterize alteration of the neuronal marker NAA in schizophrenia and to develop imaging of the inhibitory transmitter GABA. A milestone was achieved this year with the first successful measurement of prefrontal GABA tissue concentration by Drs. Kegeles, Shungu, and Hetterington. These studies were obtained on the 4T magnet at Brookhaven National Laboratory. This technique should play a major role in the future in unraveling alterations in GABA system in the prefrontal cortex in patients with schizophrenia, and with mood and anxiety disorder. The future availability of several high field magnets on our campus, including one at PI, will enable the use of this technique for clinical studies conducted here.

Functional Magnetic Resonance Imaging (fMRI) Program The successful recruitment of Eric Zarahn, Ph.D. in February 2000 enabled us to initiate fMRI studies in clinical conditions. Dr. Zarahn provided the expertise needed for the complex issue of statistical analysis of the fMRI signal. In collaboration with Drs. De la Paz, Lombardo, and Zarahn studies were initiated of prefrontal cortex activations in patients with schizophrenia while they were performing working memory tasks. These studies will provide functional data that can then be correlated with the neurochemical measurements obtained on the same patients with PET and MRS.