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WO1998048785A2 - Diagnostic d'un syndrome d'insatisfaction a l'aide de polygene allelique et traitement associe - Google Patents

Diagnostic d'un syndrome d'insatisfaction a l'aide de polygene allelique et traitement associe Download PDF

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WO1998048785A2
WO1998048785A2 PCT/US1998/008684 US9808684W WO9848785A2 WO 1998048785 A2 WO1998048785 A2 WO 1998048785A2 US 9808684 W US9808684 W US 9808684W WO 9848785 A2 WO9848785 A2 WO 9848785A2
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Prior art keywords
allele
gene
genes
adhd
rds
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WO1998048785A3 (fr
WO1998048785A9 (fr
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Kenneth Blum
David E. Comings
John L. Ivy
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KENNETH BLUM Inc
City of Hope National Medical Center
University of Texas System
University of Texas at Austin
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KENNETH BLUM Inc
City of Hope National Medical Center
University of Texas System
University of Texas at Austin
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Priority to AU72677/98A priority Critical patent/AU7267798A/en
Priority to EP98920019A priority patent/EP0979092A2/fr
Priority to IL13263498A priority patent/IL132634A0/xx
Priority to JP54736498A priority patent/JP2002511850A/ja
Priority to CA002288990A priority patent/CA2288990A1/fr
Application filed by KENNETH BLUM Inc, City of Hope National Medical Center, University of Texas System, University of Texas at Austin filed Critical KENNETH BLUM Inc
Publication of WO1998048785A2 publication Critical patent/WO1998048785A2/fr
Publication of WO1998048785A3 publication Critical patent/WO1998048785A3/fr
Publication of WO1998048785A9 publication Critical patent/WO1998048785A9/fr
Priority to IS5233A priority patent/IS5233A/is
Priority to NO995257A priority patent/NO995257L/no
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/465—Nicotine; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention in part, relates to the coupling of certain anti-craving compositions and specific genotyping of a number of genes all involved in neurotransmitter function of reward behavior.
  • An aspect of this invention is the understanding of the involvement of how certain established neurotransmitters work in concert to activate neuropathways in the meso-limbic system of the brain leading to feelings of well being, and the development of compositions that affect these neuropathways.
  • This invention in part, relates to the utilization of precursor amino acids and certain herbal compounds to enhance attentional processing and memory as well increase focus in healthy individuals, as well as to enhance weight loss and control overeating.
  • diagnostic methods of neurological disorders and behaviors utilizing genetic polymorphisms of neurotransmitter genes, and therapeutic methods of treatment of patients so identified using the compositions ofthe invention. Also disclosed are diagnostic methods for polygenic traits.
  • the mesolimbic dopamine system connects structures high in the brain, especially the orbiofrontal cortex (in the prefrontal area behind the forehead) with the amygdala in the brain's center, and with the nucleus accumbens, which has been proven in animal research to be a major site of activity in addiction.
  • the various brain pathways involved in multiple addictions converge on certain dopaminergic receptors (DI, D2, D3, D4, D5) where the D2 site seems to be most prominent.
  • dopamine is released in the nucleus accumbens and the hippocampus (Koob and Bloom, 1988). Dopamine appears to be the primary neurotransmitter of reward at these reinforcement sites.
  • NA and the locus coeruleus (LC) play a role in arousal and vigilance, critical aspects of attention (Aston-Jones et al, 1984). It has been proposed that stress tolerance and good performance on tasks were related to low basal or tonic levels of catecholamines and to higher acute releases during mental stress (Traditionally, 1989). The opposite may occur in ADHD, with an increased baseline tonic stimulation of NA and a decreased release of catecholamines during stress (Pliszka et al, 1996).
  • MHPG 3-methoxy-4-hydroxyphenylglycol
  • d-amphetamine and desipramine both of which are commonly used in the treatment of ADHD, lead to a significant decrease in the excretion of MHPG has been proposed (Mefford and Potter, 1989; Shekim et al, 1979).
  • methylphenidate (Ritalin) the most commonly prescribed medication for the treatment of ADHD does not result in a decrease in MHPG excretion (Zametkin et al, 1985) and other medications that reduce MHPG excretion, such as fenfluramine (Donnelly et al, 1989), are not effective in the treatment of ADHD.
  • NA and adrenergic ⁇ 2-receptors played a role in some forms of ADHD through a dysregulation at the LC of the posterior cortical attention system (Posner and Peterson, 1990; Pliszka 1996) of the parietal/temporal lobes, and that a second form of ADHD was due to dopaminergic defects that primarily affected the prefrontal lobe attentional system which was associated with impulsivity and disorders of executive dysfunction.
  • D2 receptor Comings et al, 1991
  • D4 dopamine D 4 receptor
  • DAT dopamine transporter
  • ADHD + cognitive disorders was due to a dysregulation of NA metabolism of the LC involving adrenergic ⁇ 2 receptors, and primarily affected the posterior attention system ofthe parietal cortex (Halperin et al, YEAR) Since these brain areas are in proximity to auditory and linguistic processing regions, this could account for the comorbid cognitive disabilities. It would be a mistake to assume that these are pure forms since ADHD is a polygenic disorder (Comings et al, 1996), and most individuals are likely to have inherited genes for both types. Studies in primates show that NA and defects in adrenergic ⁇ 2 receptors also play a role in prefrontal lobe cognitive defects (Arnsten, 1997).
  • neurotransmitters involved in the biology of reward is complex, at least three other neurotransmitters are known to be involved at several sites in the brain: serotonin in the hypothalamus, the enkephalins (opioid peptides) in the ventral tegmental area and the nucleus accumbens, and the inhibitory neurotransmitter GABA in the Substantia nigra, ventral tegmental area and the nucleus accumbens (Stein and Belluzzi, 1986; Blum and Kozlowski, 1990).
  • GABA GABA
  • the glucose receptor is an important link between the serotonergic system and the opioid peptides in the hypothalamus.
  • An alternative reward pathway involves the release of norepinephrine in the hippocampus from neuronal fibers that originate in the locus coeruleus.
  • alcohol preferring rats When compared to normal non-alcohol preferring rats, alcohol preferring rats have fewer serotonin neurons in the hypothalamus, higher levels of enkephalins in the hypothalamus (because less is released), more GABA neurons in the nucleus accumbens and a lower density of D2 receptors in certain areas of the limbic system (McBride et al, 1995; Smith et al, 1997; and McBride et al, 1997).
  • Androgent Receptor gene Specific mutations of the AR gene have been reported to cause a wide range of types of androgen insensitivity syndromes (Gott Kunststoff et al, 1977).
  • two sets of polymorphic tricnucleotide repeat sequences, CAG (Edwards et al, 1992) and GGC (Sleddens et al, 1993; Sleddens et al, 1992), resulting in polyamino acid tracts in the protein, are present in the first exon of the AR gene.
  • CAG trinucleotide repeat When highly expanded, from 43 to 65 times, the CAG trinucleotide repeat has been shown to cause X-linked spinal muscular atrophy (La Spada et al, 1991).
  • the repeat length in the normal population is 11 to 31 times (Edwards et al, 1992).
  • the non-highly expanded alleles of micro- and minisatellites present in the normal population might play a direct role in the regulation of genes. This was based on the observation that most short tandem repeats are associated with the formation of Z-DNA (Schroth et al, 1992), and Z-DNA has repeatedly been implicated in various aspects of gene regulation (Rich et al, 1984; Hamada et al, 1982; Wolff et al, 1996). Since the amount of Z-DNA formed is highly sensitive to the length of the repeats (Schroth et al, 1992), it was suggested that the size of the repeat alleles could themselves be related to phenotypic effects (Comings, 1997).
  • the opposing effect of the Dj and D 2 receptor agonists on cocaine seeking behavior in rats have been reported (Self et al, 1996).
  • TD probands, smokers, and pathological gamblers were consistent with negative heterosis, in that the most consistent difference was a relative decrease in the frequency of 12 heterozygotes and an increase in 11 and 22 homozygotes of the Dde 1 polymorphism (Comings et al. (1996)).
  • positive heterosis was present at the DRD2 gene, with quantitative scores being highest for 12 heterozygotes and lowest for 11 and 22 homozygotes. While the results for ADHD at the DRDl locus alone was not significant, there was a significant additive effect of examining the presence of negative heterosis at the DRDl gene and/or positive heterosis at the DRD2 gene (Comings et al, 1997k).
  • D2A1 Dopamine D2 Receptor Gene
  • the Taql D2 Al carriers showed a significantly lower relative glucose metabolism in the putamen, nucleus accumbens, frontal and temporal gyri and medial prefrontal, occipito-temporal and orbital cortices than those with the A22 genotype (Nobel et al, 1997).
  • the Taql D2 Al carriers had a significantly decreased dopamine D2 receptor B max in the basal ganglia (Noble et al, 1991k).
  • Enkephalin increases blood flow in similar regions as methylphenidate and may therefore involve a dopaminergic mechanism (Blum et al, 1985k).
  • a significant decrease in dopamine D2 receptor density was measured in individuals with detachment, social isolation, and lack of intimate friendships (Farde et al, 1997k).
  • the DRD 2 gene A allele has been found to associate with a number of behaviors including severe alcoholism, polysubstance dependence, crack/cocaine addiction, tobacco smoking, pathological gambling, lack of a major depressive episode, and carbohydrate bingeing or generalized to DSM- IV substance use disorder (Blum et al, 1996e; Blum et al, 1995b; Comings et al, 1996c).
  • the MCMI-II assessed schizoid/avoidant cluster compared to other Axis II diagnostic clusters (antisocial, narcissistic, paranoid) significantly correlated with alcohol abuse scales (Corbisiero et al, 1991).
  • CSF HVA levels and the DRD2 Taql Al I 2 polymorphism were examined in Finnish and American alcoholics, and no association was found when examining the 1 vs. 2 alleles, and not the 1 ,1+1,2 vs. the 2,2 genotypes (Goldman et al, 1992).
  • Heterosis at the DRD2 gene was indicated by comparison of the CSF levels of HVA, to the DRD2 genotype using Taql polymorphism (Jonsson et al, 1996k).
  • the 12 heterozygotes showed the highest inattention score subjects who were 12 heterozygotes had the lowest levels of CSF HVA (Jonsson et al, 1996).
  • the highest levels of HVA were seen in the 11 homozygotes, with the 22 homozygotes being intermediate.
  • ADHD probands showed a significant association with the 48bp variant of the D4 gene, but not the DRD2, DRD3 or the serotonin transporter genes.
  • the 7-fold repeat allele of the DRD4 occurred significantly more frequently in that children with ADHD.
  • novelty seeking characterized as impulsive, exploratory, fickle, excitable, quick tempered and obsolete
  • DRD2 Al allele in cocaine dependent probands was associated with the opposite: low novelty seeking, characterized by reflective, rigid, stoic, slow-tempered, avoidant, as well as having enhanced withdrawal depression (Compton et al, 1996).
  • D 2 dopamine receptor (DRD2) Al allele with alcoholism and drug abuse (Blum et al, 1990).
  • Reduced central dopaminergic function has been suggested in subjects who carry the Al allele (Al + ) compared with those who do not (AT) (Nobel et al 1997).
  • the genes responsible for alcoholism are unknown, although the many studies to date indicate a significant role for the DRD2 gene in more severe cases (Noble, 1993; Blum et al, 1995).
  • the DRD2 gene has been associated with the compulsive behavior ⁇ Comings and Comings, 1987b) and addictive, impulsive behaviors, including compulsive eating, gambling and smoking. (Self et al, 1996; Ogilvie et al, 1996; Blum et al, 1995b; Blum et al, 1996e). These behaviors have previously been reported to be associated with the DRD2 gene (Comings et al, 1993a; Noble et al, 1994d; Blum et al, 1996a; Comings et al, 1996c; Noble et al, 1994c; Noble, 1993; Comings et al, 1996e) in subjects distinct from the TS group.
  • Dopamine D2 receptor availability was significantly lower in alcoholics than in nonalcoholics, and was not correlated with days since last alcohol use (Volkow et al, 1997).
  • the ratio DRD2 receptor to transporter availability was significantly higher in nonalcoholics than in alcoholics.
  • Alcoholics showed significant reductions in D2 receptors (postsynaptic marker) but not in DA transporter availability tpresynaptic marker) when compared with nonalcoholics. Because D2 receptors in striatum are mainly localized in GABA cells, these results provide evidence of GABAergic involvement in the dopaminergic abnormalities seen in alcoholics.
  • DRD3 Dopamine D 3 Receptor Gene
  • DRD4 a 48 bp and 16 amino-acid repeat polymorphism within the DNA coding for the third cytoplasmic loop responsible for binding to guanine -nucleotide proteins (Van Tol et al, 1992k; Lichter et al, 1993k) has been reported. This DNA region is repeated 2 to 1 1 times, with the most common alleles being the 2, 4, and 7 repeat. The 7 allele demonstrates a blunted response to dopamine in regards to intracellular adenyl cyclase inhibition (Asghari et al, 1995k).
  • DAT1 Dopamine Transporter Gene
  • the DAT1 gene marker frequencies at the vesicular transporter locus showed substantial heterogeneity in different Caucasian-Americans originating from different European countries, but no association with substance abuse was evident (Uhl et al, 1993; Persico et al, 1993). Distributions of the DAT1 VNTR alleles do not distinguish any substance user or control sample for psychostimulant abusers (Persico et al, 1996), however an association was observed with Japanese alcoholics (Muramatsu and Higuchi, 1995). The DAT1 gene has also been implicated as having a role in compulsive and addictive disorders.
  • Dopamine- ⁇ -Hydroxylase D ⁇ H is one of the major enzymes for dopamine metabolism and catalyzes the conversion of dopamine to norepinephrine (NE).
  • NE norepinephrine
  • the inhibition of D ⁇ H activity results in a decrease in norepinephrine levels which releases the inhibition of tyrosine hydroxylase resulting in the excessive production of dopamine.
  • the later is associated with hyperactivity, aggression, self- stimulation, and stereotypic movements (Randrup and Scheel-Kruger, 1966; Shekin et al, 1983k).
  • D ⁇ H dopamine-Beta-hydroxylase
  • Linkage studies between the D ⁇ H locus and schizophrenia (Aschauer and Meszaros, 1994), alcoholism, depression, manic- depression and Tourette's syndrome (Comings, et al, 1986) have been negative.
  • sib pair analyses suggest a weak linkage between the ABO blood group and D ⁇ H, and some psychiatric disorders such as depression and alcoholism (Wilson et al, 1992).
  • Cannabinoid Receptors While the association of cannabinoid receptors with the reward pathways may be primary, it is more likely that the effect is secondary through the modulating effect of anandaide and cannabinoid receptors on dopamine metabolism. This is consistent with the similarity between the results with CB1 receptors and the DRD2 receptors. Like the CB1 gene the association of genetic variants of the DRD2 gene with polysubstance abuse has been more reproducible (O ⁇ ara et al, 1993; Smith et al, 1992; Noble et al, 1993; Comings et al, 1994) than the association with alcoholism per se. One interpretation of these observations is that the dopaminergic-cannabinoid reward pathways are activated more by drugs, especially cocaine and amphetamines, than by alcohol (DiChiara and Imperato. 1988).
  • Activation of the mesolimbic dopamine system is known to trigger a relapse to cocaine seeking behavior in animal models of drug dependence.
  • This priming effect is enhanced by dopamine D 2 agonists but inhibited by dopamine D, agonists (Self et al 1996).
  • the ability of anandamide to cause a decrease in the ratio of D, and D 2 receptors in the striatum may be the link that accounts for the role of CB1 variants in drug dependence.
  • Tivol et al (1996) have recently sequenced the exons of 40 control males who showed a > 100-fold variation in MAO A enzyme activity. There was remarkable conservation of the coding sequence. Only five polymorphisms were found. Of these, four involved the third codon position with no change in the amino acid sequence. The other was a neutral lys — arg substitution.
  • Nicotine Receptor Genes The gene for the CHRNA4 gene is located on chromosome 20ql3.2-13.3 (Steinlein et al, 1994) and consists of 6 exons over 17 kb of genomic DNA (Steinlein et al, 1996).
  • a Ser248Phe missense mutation in the transmembrane domain 2 of the CHRNA4 gene was found to be associated with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) in one extended
  • EEG (Steinlein et al, 1992) have also been linked to the region of the CHRNA4 locus. D20S19, a highly polymo ⁇ hic locus, is in tight linkage with the genes for all three of these disorders (Steinlein et al, 1996).
  • VNTR polymo ⁇ hism located in the first intron of the CHRNA4 gene was reported by Weiland and Steinlein (Weiland and Steinlein).
  • Ser248Phe missense mutation associated with ADNFLE and four silent polymo ⁇ hisms, Steinlein et al, Ser248Phe missense mutation reported a modest increase in the frequency of the T allele of the Cfol 595 polymo ⁇ hism in common idiopathic generalized epilepsies of childhood (.085) versus controls (.027).
  • Micro/minisatellite polymorphisms Studies of behavioral phenotypes associated with micro/minisatellite polymo ⁇ hisms at different neuropsychiatric candidate genes have found a significant association between the shorter or longer alleles with various quantitative behavioral traits and mini- or microsatellites at the following genes: MAOA, MAOB, HTR1A, DATl, DRD4, HRAS, HTT, OB, CNR1, GABRA3, GABRB3, FRAJXA, and NO (Comings et al, 1996k; Comings et al, 19961; Comings et al, 1996m; Johnson et al. 1997; Comings et al, 1998; Gade et al, 1997).
  • Taql polymorhisms of the apolipoprotein gene ⁇ APOE-D was found to associate with obese subjects and between the APO-D and fasting-insulin. This work suggests that that the APO-D polymo ⁇ hism may be a genetic marker for both obesity and hyperinsulinemia (Vijayaraghavan et al, 1994).
  • catecholamine innervation of the cerebral cortex may be the control of attention.
  • catecholamine projections to the cerebral cortex from the reticular formation namely dopamine neurons in the ventral tegmentum of the midbrain and the NE neurons of the locus coeruleus in the upper pons.
  • ACH acetylcholinergic
  • DA dopaminergic systems
  • Choice accuracy deficit induced by blockade of either muscarinic- or nicotinic-ACH receptors The choice accuracy deficit induced by blockade of muscarinic receptors with scopolamine can be reversed by the dopamine receptor blocker, haloperidol.
  • the specific DAD1 blocker SCH23390 also has this effect, whereas the specific D 2 blocker raclopride did not. This effect is seen with the D 2 antagonist raclopride, but not with the Dj antagonist SCH23390.
  • the D 2 receptor was indicated in nicotinic actions on cognitive function by the finding that the selective D 2 agonist LY 1771555 reverses the choice accuracy deficit caused by mecamylamine.
  • the effectiveness of these selective DA treatments in reversing cognitive deficits was due to ACH under-activation (Levin et al. 1990k).
  • NE Norepinephrine
  • LC coeruleo- cortical
  • SP neurokinin substance P
  • N-terminal SP1-7 enhanced memory
  • C-hepta- and hexapeptidsequences of SP proved to be reinforcing in a dose equimolar to SP.
  • bromocriptine a D 2 dopamine receptor agonist
  • This form of memory in which some aspect of a stimulus is maintained over a short interval of time, has also been found to be closely tied to prefrontal cortical function in both lesion and single unit recording studies with monkeys and in neuro-imaging studies in humans (Goldman et al, 1987k; Jonidas et al, 1993).
  • a selective positive effect of bromocryptine, in reducing release rates in alcoholics as a function of dopamine D 2 receptor genotype has also been reported.
  • a double-blind study demonstrated that a D 2 agonist bromocryptine or a placebo administered to alcoholics who were carriers of the Al allele (Al/Nl and A1/A2 genotypes), or who only carried the A2 allele (A2/A2) reduced craving and anxiety among the Al carriers who were treated with bromocryptine.
  • the attrition rate was highest among the Al carriers who were treated with placebo.
  • the bromocryptine effect on the Al carriers was much more robust as one approached the six wk period of treatment.
  • Dopamine D 2 agonist bromocryptine can improve higher- level cognitive functions. Studies using sophisticated techniques in animals, including microdialysis measurements, have demonstrated changes in neurotransmitter output following precursor amino acid loading (Hernandez et al, 1988).
  • phenylalanine deficiency PHD
  • phenylalanine and tyrosine constitute the two initial steps in the biosynthesis of dopamine, which, in its turn, is the metabolic precursor of NE.
  • the extracellular phenylalanine concentration influences brain function in PHD by decreased dopamine synthesis. It has been shown to induce EEG slowing and has prolonged the performance time on neuropsychological tests.
  • the tyrosine concentration in the CNS is reduced in PHD, possibly implying an insufficient substrate of tyrosine for catecholamine synthesis due to competition inhibition, for instance across the blood brain barrier.
  • a combination of precursor amino acids having enkephalinase inhibition activity may be used for the treatment of cocaine dependence (U. S. Patent No. 5,189,064). It is known that acute use of cocaine can improve certain aspects of brain electrophysiological dysfunction (Maurer et al, 1988k). Chronic cocaine abuse alters attentional processing (Noldy et al, 1990k). It is known that acute use of cocaine can improve certain aspects of brain electrophysiological dysfunction (Jonsson et al, 1996). However, paradoxically, chronic cocaine abuse alters attentional processing (Braverman and Blum, 1996). Although still controversial, attentional processing has been shown to be dependent on biogenic amine regulation (Lyoo et al, 1996).
  • Obesity and Neurological Functions Obesity generally is defined as being 20% or more over ideal body weight. Numerous methods of weight reduction have been attempted including hypocaloric balanced diets, "fad” diets, behavior modification, drugs ⁇ i.e. D-phenflouramine, phenteramine, etc.), surgery, total starvation, jaw wiring, and combinations of these methods. Most of these are short- term approaches to the problem and have been only transiently effective and some can even pose serious danger (Lockwood and Amatruda, 1984). Even if weight loss is demonstrated in the short-term, the weight usually is regained following discontinuation of the weight-loss regiment. Despite the fact that about 28% of the American population is obese, obesity is widely perceived as a food-addiction, a self- imposed condition with cosmetic rather than health indications (Krai et al, 1989; Weintraub and Bray, 1989).
  • D2 receptor D2
  • DATl dopamine transporter
  • the primary neurotransmitters involved in eating behavior include the monoamines dopamine (DA), norepinephrine (Ne), epinephrine (EPI), and serotonin (5-HT); the inhibitory neurotransmitter gamma-aminobutyric (GABA); and a variety of neuropeptides such as the pancreatic polypeptides, opioid peptides, hormone-releasing factors, and various gut-brain peptides (for reviews see Cooper et al, 1988; Gosnell, 1987; Bouchard, 1994).
  • DA dopamine
  • Ne norepinephrine
  • EPI epinephrine
  • serotonin 5-HT
  • GABA inhibitory neurotransmitter gamma-aminobutyric
  • neuropeptides such as the pancreatic polypeptides, opioid peptides, hormone-releasing factors, and various gut-brain peptides (for reviews see Cooper et al, 1988; Gosnell, 1987;
  • Nicotine Nicotine also releases dopamine, and nicotine has been found to improve memory performance in a variety of tests in rats, monkeys, and humans (DiChiara et al, 1988). Nicotine in a dose dependent fashion reduced incorrect responding on discrimination behavior in rats (Geller et al, 1970). This effect was similar to chlordiazepoxide but could not be mimicked by the stimulant caffeine (Geller et al, 1970).
  • Nicotine in the form of gum or skin patches (Sanberg et al, 1988; McConville et al, 1992; Sanberg et al, 1997) has been shown to be effective in the treatment of tics in some subjects with Tourette syndrome (TS), and cigarette smoking has been reported to enhance attention, arousal, learning and memory (Wesnes and Warburton, 1984; Warburton, 1992; Balfour and Fagerstr ⁇ m, 1996) and to improve the symptoms of ADHD (Coger et al, 1996; Conners et al, 1996; Levin et al, 1996).
  • the working memory deficit caused by the nicotinic antagonist mecamylamine was potentiated by the D1/D2 DA antagonists haloperidol and the specific D 2 antagonist raclopride.
  • the mecamylamine- induced deficit was reversed by co-administration of the D2/D3 agonist quinpirole.
  • Nicotine also has significant interactions with dopamine drugs with regard to working memory performance in the radial-arm maze.
  • the dopamine agonist pergolide did not by itself improve radial-arm choice accuracy. Nicotine was effective in reversing this deficit.
  • the D2/D3 agonist quinpirole improved RAM choice accuracy relative to either drug alone.
  • Acute local infusion of mecamylamine to the midbrain dopamine nuclei effectively impairs working memory function in the radial-arm (Noble et al, 1998).
  • Chromium Salts Trivalent chromium is a mineral essential for normal insulin function (Jeejeehboy et al, 1977; Schwartz et al, 1959).
  • CAD coronary artery disease
  • NIDDM non-insulin- dependent diabetes mellitus
  • Chromium Picolinate (CrP) is the most heavily used, studied and promoted chromium compound, but in vitro work suggests that chromium nicotinate may be also viable in the area of weight loss and changes in body composition.
  • Previous research has shown chromium picolinate supplementation decreasing fat mass and increasing fat- free mass (Kaats et al, 1991 ; Page et al, 1991). Pervious studies of exercise training have shown increases in fat free mass as well (Stefanick, 1993).
  • Nutritional Supplements in Treatment of Behavioral Disorders may underlay a variety of psychiatric and behavioral disorders (Blum et al, 1996c; Persico and Uhl, 1997; Noble et al, 1991). Specifically, anomalous regulation of dopamine, serotonin, norepinephrine, gammaminobutyric acid (GABA), glutamine, and the opioid peptides are thought to play crucial roles in the addictive disorders, particularly those involving alcohol and cocaine abuse (Pohjalainen et al, 1996). Consequently, these observations have provided momentum to the idea that ingestion of selected nutrients could affect mood and therefore behavior in humans.
  • GABA gammaminobutyric acid
  • the invention first provides a composition for the treatment of Reward Deficiency Syndrome (RDS) behaviors in a subject.
  • this composition includes at least one of the following components: an opiate destruction- inhibiting amount of at least one substance which inhibits the enzymatic destruction of a neuropeptidyl opiate, the substance being either amino acids, peptides.
  • a neurotransmitter synthesis-promoting amount of at least one neurotransmitter precursor the neurotransmitter precursor being either a dopamine precursor such as L-Tyr, L-Phe and L-dopa, a serotonin precursor such as L-T ⁇ and 5-hydroxytryptophan, or a gamma amino butyric acid (GABA) precursor such as L-glutamine, L-glutamic acid, and L-glutamate; a tryptophan concentration enhancing amount of chromium picolinate or chromium nicotinate; a compound that releases enkephaline, the enkephaline releaser being, but not limited to, a peptide, and preferably a D-amino acid containing peptide; or an opiate antagonist amount of at least one compound which blocks the effects of an opiate at either the delta, mu, kappa, sigma, or epsilon receptors.
  • GABA gamma amino butyric acid
  • the composition is used in preventing or reducing a subject's unwanted weight.
  • the composition is preferably used in the treatment of Attention Deficits disorder, attentional processing or memory.
  • the composition more preferably includes a neurotransmitter synthesis-promoting amount of at least one neurotransmitter synthesis promoting substance selected from the group Rhodila or hubazine.
  • a neurotransmitter synthesis-promoting amount of at least one neurotransmitter synthesis promoting substance selected from the group Rhodila or hubazine.
  • derivative may refer to a chemically modified compound, and “analog” refers to a different compound that is similar properties or structure to the compound it is being compared.
  • this composition may be used in the treatment of all RDS related behaviors disclosed herein.
  • RDS behaviors are those behaviors related to a chemical imbalance manifests itself as one or more behavioral disorders related to an individual's feeling of well-being with anxiety, anger or a craving for a substance.
  • RDS behaviors include, alcoholism, SUD, smoking, BMI or obesity, pathological gambling, carbohydrate bingeing, axis 1 1 diagnosis, SAB, ADD/ADHD, CD, TS, family history of SUD, and Obesity, are described herein.
  • the invention also provides a method of treating a subject for RDS behaviors, including but not limited to SUD, Obesity, Smoking, Tourettes Syndrome, ADHD, Schizoid/Avoidant Behavior, Aggression, Posttraumatic stress syndrome, PMS or tobacco use.
  • RDS behaviors are not specifically limited to these disorders, as many types of sub-disorders are encompassed by these conditions.
  • attention deficit hyperactivity disorder may manifest itself as alcohol, drugs, obsessive compulsive behaviors, learning disorders, reading problems, gambling, manic symptoms, phobias, panic attacks, oppositional defiant behavior, conduct disorder, academic problems in grade school, smoking, sexual behaviors, schizoid, somatization, depression, sleep disorders, general anxiety, stuttering, and tics disorders. All these behaviors, and others described herein as associated with RDS behaviors or genes involved in the neurological pathways related to RDS, are included as RDS behaviors as part of this invention.
  • Sleep Disorders including Primary Sleep Disorders such as Dyssomnias which include Primary Insomnia 307.42, Primary Hypersomnia 307.44, Narcolepsy 347, Circadian Rhythm Sleep Disorder, 307.45, Dyssomnia NOS 307.47, Parasomnias which include Nightmare Disorder 307.47, Sleep Terror Disorder 307.46, Sleepwalking Disorder 307.46, Parasomnia NOS 307.47, Sleep Disorders Related to Another Mental Disorder which include
  • Cannabis-Induced Psychotic Disorder with hallucinations 292.12, Cannabis- Induced Anxiety Disorder, 292.89, Cannabis Related Disorder NOS, 292.9, Cannabis Intoxication, 292.89, Cocaine Related Disorders which include Cocaine Dependence, 304.20, Cocaine Abuse, 305.60, Cocaine Intoxication. 292.89, Cocaine Withdrawal, 292.0, Cocaine Intoxication Delirium, 292.81 , Cocaine-Induced Psychotic Disorder with delusions, 292.1 1, Cocaine-Induced Psychotic Disorders with hallucinations,
  • Opioid Abuse 305.50, Opioid Intoxication, 292.89, Opioid Intoxication Delirium, 292.81, Opioid-Induced Psychotic Disorder, with delusions, 292.11, Opioid-Induced Psychotic Disorder with hallucinations, 292.12, Opioid-Induced Anxiety Disorder, 292.89, Opioid Related Disorder NOS, 292.9, Opioid Intoxication, 292.89, Opioid Withdrawal, 292.0; Polysubstance Related Disorders which include Polysubstance Dependence, 304.80; Tic Disorders which include Tourette's Disorder, 307.23, Chronic Motor or Vocal Tic Disorder 307.22, Transient Tic Disorder 307.21, Tic Disorder NOS 307.20, Stuttering 307.0, Autistic Disorder, 299.00, and Somatization Disorder 300.81.
  • RDS disorders are defined as would be known to one of skill in the art, such as Novelty Seeking, defined in (Clonigen et al, 1993).
  • Other disorders, if not specifically defined herein, are the same as commonly known to one of skill in the art, including common abbreviations.
  • the amount of each of the above mentioned compounds administered daily for use in the treatment of RDS behaviors or disorders may be of about 1, about 2, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 1, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24. about 25, about 26, about 27, about 28, about 29, about 30, about 31 , about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40.
  • the preferred ranges and components of the composition are 460 mg DL- phenylalanine, 25 mg L-tryptophan, 25 mg L-glutamine, and 5 mg pyridoxal -5'- phosphate administered daily.
  • the subject is tested using the methods disclosed herein or known to one of skill in the art to determine whether the subject has a family history of chemical dependency, wherein the family history indicates an improved likelihood for successful treatment.
  • the treatment inhibits binge eating.
  • the treatment inhibits craving.
  • the composition contains chromium salts.
  • a subject is tested using a molecular biology assay, as described herein, for an allele associated with an RDS or psychological behavior, and the presence of such an diagnostic allele is indicative a subject more likely to respond positively to the compositions disclosed herein for therapy.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein for the presence of at least one ofthe following alleles: D 2 Taql Al, BI, Cl or exon " haplotype HTR2A - C allele homozygous OB - homozygosity for ⁇ 208 BP alleles of 1875 dinucleotide repeat polymo ⁇ hism human chromosome 2 microsatellite polymo ⁇ hism, APO-D - Taql 2.2 or 2.7 BP, or OB gene D7S1875, where detection of the above mentioned alleles indicates an improved likelihood for a successful response to the treatment.
  • alleles D 2 Taql Al, BI, Cl or exon " haplotype HTR2A - C allele homozygous OB - homozygosity for ⁇ 208 BP alleles of 1875 dinucleotide repeat polymo ⁇ hism human chromosome 2 microsatellite
  • the composition includes an effective amount of chromium nicotinate, and the subject is tested for the presence of the DRD2 Al allele, wherein the presence of the DRD2 Al allele indicates an improved likelihood of response using the treatment.
  • the composition includes an effective amount of chromium picolinate, and the subject is tested for the presence of the DRD2 A2 allele, wherein the presence of the DRD2 Al allele indicates an improved likelihood of response using the treatment.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein for the presence of at least one of the following alleles: DI (homozygosity of Dde Al) D2 ⁇ Taql Al) D4 (VNTR 2) D5 (dinucleotide 13 alleles range 135-159 BP) DATl VNTR (10/10) D ⁇ H ⁇ Taql BI allele), where detection of the above mentioned alleles indicates an improved likelihood for a successful response to the treatment.
  • DI homozygosity of Dde Al
  • D2 ⁇ Taql Al) D4 (VNTR 2)
  • D5 dinucleotide 13 alleles range 135-159 BP)
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein for the presence of at least one of the following alleles: DI (homozygosity of Dde Al) D2 ⁇ Taql 1) D4 (VNTR 2) D5 (dinucleotide 13 alleles range 135-159 BP) DATl VNTR (10/10) D ⁇ H ⁇ Taql BI allele) MAOA(X), where detection of the above mentioned alleles indicates an improved likelihood for a successful response to the treatment.
  • the composition includes an effective amount of Rhodila or hubazine.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein for the presence of at least one of the following alleles DI (homozygosity of Dde Al) D2 (Taql Al, BI, Cl), where detection of the above mentioned alleles indicates an improved likelihood for a successful response to the treatment.
  • DI homozygosity of Dde Al
  • D2 Taql Al, BI, Cl
  • RDS behavior further comprises pathological violence, Schizoid/ Avoidant (SAB), Aggression, Anger,
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein for the presence of at least one of the following alleles D2 ⁇ Taql Al, BI, Cl, exon 6"7 ) DATl (VNTR 10/10) mNOSIa - homozygosity for ⁇ 201 BP, where detection of the above mentioned alleles indicates an improved likelihood for a successful response to the treatment.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein for the presence of at least one of the alleles from the DATl VNTR (10/10) D 2 Taql Al, BI, Cl, exon 6"7 haplotype, or alleles from the DRDl, DRD2, DRD4, HTT, HTRIA, TDO2, D ⁇ H, MAO, COMT, GABRAB, GABRB3, PENk, ADRA2A or ADRN2C genes, where detection of the above mentioned alleles indicates an improved likelihood for a successful response to the treatment.
  • the invention further provides a method of determining a genetic predisposition of a subject to at least one RDS behavior, by detecting at least one allele from the group including, but not limited to the DRDl, DRD2, DRD3, DRD4, DRD5, DATl, HTT, HTRIA, TD02, DBH, ADRA2A, ADRA2C, NET, MAOA, COMT, GABRA3, GABRB3, CNRl, CNRA4, NMDAR1, PENK, AR, CRF, HTRlD , HTR2A, HTR2C, interferon- ⁇ , CD8A, or RS7 genes, where the allele is diagnostic for an RDS behavior.
  • the allele is diagnostic for an RDS behavior.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of at least one is a V ⁇ TR polymo ⁇ hism of a MAOA gene allele, wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to an RDS behavior, including mania, OCD, sexual, sleep, grade school behavior, gambling, learning, inattention, ADHD, ADDR, impulsivity, MDE, CD, hyperactivity, phobia, schizoid behavior, general anxiety, somatization, drugs, IV drugs, read, ODD, tics, alcohol, or tobacco use.
  • an RDS behavior including mania, OCD, sexual, sleep, grade school behavior, gambling, learning, inattention, ADHD, ADDR, impulsivity, MDE, CD, hyperactivity, phobia, schizoid behavior, general anxiety, somatization, drugs, IV drugs, read, ODD, tics, alcohol, or tobacco use.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of at least one DRD 2 gene A, allele, the DAT gene, V ⁇ TR 10/10 allele, or the D ⁇ H gene B, allele, wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to an RDS behavior, including schizoid or Avoidant.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of an increased number of (NAT) n triplet repeats in the CNRl gene, wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to an RDS behavior, including Drug Use.
  • NAT molecular biological assay
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of an increased number of the D7S1873, D7S1875, D7S514 or D7S680 dinucleotide repeats in the OB gene, wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to an RDS behavior including obesity, anxiety, depression, psychoses, hostility, paranoid ideation, obsessive-compulsive, symptom total, general symptom index, novelty seeking, overall total, neurotogni and conscientiousness.
  • an RDS behavior including obesity, anxiety, depression, psychoses, hostility, paranoid ideation, obsessive-compulsive, symptom total, general symptom index, novelty seeking, overall total, neurotognitiveness.
  • the allele is the D7S1875 dinucleotide repeats is greater than 225 bp in length, and this allele is present in both copies of the CNR/ gene.
  • another allele detected is the D2AI allele of the DRD2 gene.
  • the RDS behavior is obesity.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of the D2AI allele of the DRD2 gene, wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to an RDS behavior, including Tourette's Syndrome, manic symptoms, oppositional defiant, sexual, ADHD-R, schizoid, ADHD, tics, major depression, conduct, stuttering, obsessive-compulsive, somatization, alcohol abuse, learning, and sleep problems.
  • a genetic predisposition to an RDS behavior including Tourette's Syndrome, manic symptoms, oppositional defiant, sexual, ADHD-R, schizoid, ADHD, tics, major depression, conduct, stuttering, obsessive-compulsive, somatization, alcohol abuse, learning, and sleep problems.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of the Taq Al allele of the D ⁇ H gene, wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to an RDS behavior, including Tourette's Syndrome, ADHD, smoking, learn, grade school, ADHD-R, oppositional defiant, tics, mania, alcohol, reading, drug abuse, sleep, stuttering, obsessive compulsive, somatization and major depression.
  • the alleles detected are the Taq BI allele and the Taq Al allele of the D ⁇ H gene, and that the RDS behavior is Tourette's Syndrome.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of the 10 allele of the DATl gene, wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to an Tourette's syndrome, autism, somatization, alcohol, AD ⁇ D-R, major depression, panic, obsessive compulsive, general anxiety, mania, oppositional defiant, sexual, read, and ADHD.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of the 10 allele of the DATl gene, the Taq Al allele of the D ⁇ H gene, or the D 2 A1 allele of the DRD2 gene, wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to ADHD, stuttering, ADHD-R, oppositional, defiant, tics, conduct, obsessive compulsive, mania, alcohol, general anxiety, panic schizoid, sleep, sexual, drugs, and major depression.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of the Ddel allele of the DRDl gene, wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to alcohol, smoking, compulsive eating, tics, gambling, drugs, reading, shopping, oppositional defiant, major depressive episode, schizoid, ADHD, conduct disorder, obsessive compulsive, and mania.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of the Taql Al and the Taql A2 alleles of the DRD2 gene, wherein the presence of those alleles are diagnostic for a subject with a genetic predisposition to oppositional defiant, conduct disorder, eating, smoking, gambling, ADHD, obsessive compulsive, mania, and alcohol.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of the 1 1 or the 22 genotype of the DRDl gene, wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to Tourettes syndrome, smoking, and gambling.
  • the alleles detected are two copies per genome ofthe Ddel allele ofthe DRDl gene.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of the 1 1 genotype of the DRDl gene, wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to oppositional defiant behavior, conduct disorder, compulsive eating, smoking, gambling, ADHD mania, stuttering, obsessive-compulsive, and schizoid behaviors.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of the Ddel allele of the DRDl gene, wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to comprising gambling, smoking, compulsive eating, oppositional defiant, major depressive episode, ADHD, conduct disorder, schizoid, obsessive-compulsive, mania, and alcohol.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of the 1 1 or the 22 genotype of the DRDl gene, wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to comprising alcohol, smoking, compulsive eating, tics, gambling, drugs, reading, shopping, gambling, and grade school problems.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of the intron 6 G ⁇ A polymo ⁇ hism of the Tryptophan 2,3 dioxygenase gene, wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to comprising Tourettes Syndrome.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of the intron 6 G— T polymo ⁇ hism of the Tryptophan 2,3 dioxygenase gene, wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to comprising ADHD, alcohol dependence, drug dependence, pathological gambling.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of the intron 6 DGGE polymo ⁇ hism of the Tryptophan 2,3 dioxygenase gene, wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to comprising ADHD, alcohol dependence, drug dependence, pathological gambling, and depression.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of the low base pair alleles ( ⁇ 181 bp) polymo ⁇ hism of the ADRA2C dinucleotide repeat polymo ⁇ hism, wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to comprising drug use.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of the two high base pair alleles for the >183 bp of the ADRA2C dinucleotide repeat polymo ⁇ hism, wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to comprising alcohol use.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of the two homologous alleles for the presenilin-1 (PSI) polymo ⁇ hism. wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to comprising alcohol and tobacco use.
  • PSI presenilin-1
  • the alleles detected are two homologous alleles of greater than 80 bp of the CA dinucleotide repeat polymo ⁇ hism ofthe PENK gene.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of short GGC alleles of the AR gene, wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to comprising CD, ODD, or hyperactivity.
  • the subject is genetically tested, or tested by a molecular biological assay, using the methods disclosed herein, for the presence of the DRD2 allele, wherein the presence of the allele is diagnostic for a subject with a genetic predisposition to comprising Type B behavior in alcoholics, cocaine addicts, or RDS probands.
  • the invention further provides a method for determining a genetic predisposition to a polygenic trait comprising detecting at least one allele associated from the group comprising the DRDl, DRD2, DRD5, DATl, HTT, HTRIA, TD02, DBH, ADRA2A.
  • the polygenic trait is ADHD, and the allele is associated with the DRDl, DRD2, DRD5, DATl, HTT, HTRIA, TD02, DBH, ADRA2A, ADRA2C, NET, MAOA, COMT, GABRA3, GABRB3, CNRl, CNRA4, NMDARl, PENK, AR, or CRF genes.
  • the polygenic trait is a lack of susceptibility to ADHD, and the allele is associated with the DRD3, DRD4, HTRJDfi, HTR2A, HTR2C, interferon- ⁇ , CD8A, or RS7 genes.
  • the polygenic trait is OOD, and the allele is associated with the DRDl, DRD2, DRD3, DATl, HTT, HTRIA, HTR2A, HTR2C, DBH, ADRA2A, ADRA2C, MAOA, GABRA3, GABRB3, CNRl, CHRNA4, NMDARl, PENK, AR or CD8A genes.
  • the polygenic trait is tics, and the allele is associated with the DRD7, DRD5, HTRIA, HTRlD ⁇ , HTR2C, TD02, DBH, ADR2C, COMT, GABRA3, CNRl or CHRNA4 genes.
  • the polygenic trait is LD, and the allele is associated with the DRD7, HTR2C, TD02. DBH, ADR2A, ADR2C, MAOA, CNRl or CNRA4 genes.
  • the polygenic trait is LDL, and the allele is associated with the HTT, OXYR, DRD2 or PS 1 genes.
  • the polygenic trait is longevity, and the allele is associated with the PS 1 , OXYR or APOE genes.
  • the invention additionally provides a method for developing a diagnostic, polygenic assay by identifying the trait that is to be studied, creating a scale measuring the severity of the trait to be studied; selecting at least one candidate gene that may contribute to the trait, identify at least one polymo ⁇ hism associated with the candidate gene, correlating allelic patterns of the polymo ⁇ hism with the scale, and comparing the association of the allelic pattern to the correlation of the candidate gene to the trait.
  • the allelic patterns that are positively associated with the trait are added together, to form a polygenic assay that is diagnostic for a subject's susceptibility to possess polygenic trait. It is also part of the invention that allelic patterns that are negatively associated with the trait are added to form a polygenic assay that is diagnostic for a subject's lack of susceptibility to posses a polygenic trait.
  • the candidate genes include, but are not limited to, the DRDl, DRD2, DRD5, DATl, HTT, HTRIA, TD02, DBH, ADRA2A,
  • HTR1D HTR2A, HTR2C, interferon- ⁇ , CD8A, or
  • the polygenic traits include, but are not limited to ADHD, lack of ADHD, ODD, CD, LD, Tics, Drug Abuse/Dependence,
  • the polygenic assay to ADHD is the detection at least one allele associated with the DRDl, DRD2, DRD5, DATl, HTT, HTRIA, TD02, DBH, ADRA2A, ADRA2C, NET, MAOA, COMT, GABRA3, GABRB3, CNRl, CNRA4, NMDARl. PENK, AR, or CRF genes.
  • the polygenic assay is to a reduced susceptibility to ADHD comprises detecting at least one allele associated with the DRD3, DRD4, H7R/D ⁇ , HTR2A, HTR2C, interferon- ⁇ , CD8A, or RS7 genes.
  • the polygenic assay to the OOD comprises detecting at least one allele associated with the DRDl, DRD2, DRD3, DATl, HTT, HTRIA, HTR2A, HTR2C, DBH, ADRA2A, ADRA2C, MAOA, GABRA3, GABRB3, CNRl, CHRNA4, NMDARl, PENK, AR or CD8A genes.
  • the polygenic assay to the tics comprises detecting at least one allele associated with the DRDl, DRD5, HTRIA, H7R7D ⁇ , HTR2C, TD02, DBH, ADR2C, COMT, GABRA3, CNRl or CHRNA4 genes.
  • the polygenic assay to the LD comprises detecting at least one allele associated with the DRD7, HTR2C, TD02, DBH, ADR2A, ADR2C, MAOA, CNRl or CNRA4 genes.
  • polygenic assay to the elevated LDL levels comprises detecting at least one allele associated with the HTT, OXYR, DRD2, or PSI genes.
  • the polygenic assay to the longevity comprises detecting at least one allele associated with the PSI, OXYR or APOE genes.
  • the polygenic assay to the osteoarthritis further comprises detecting at least one allele associated with the COL2A1 , COL2A1, COL2A1, COL9A1, COL9A1, AGC1, IGF1, IGF1, IGF1R,
  • IGF1R IGF2, IGF2R, TGFB1, TGFB2, ILIA, IL1B, IL1R1, IL1RN, MMP9, TIMP1 or Vitamin D3 genes.
  • the word “a” or “an” or “the” may mean one or more than one.
  • FIG. 1 The DRD2 Gene in "Super Controls (Normals)” and Severe “RDS” Probands.
  • the Linear trend analysis for comparing across the groups depicted had a p ⁇ 0.000001.
  • Group I Carefully assessed for alcoholism, substance use disorder, polysubstance dependence, family history of chemical dependence and obesity nicotine dependence (smoking) BMI over 25, carbohydrate binging, autism,
  • Group VI C6
  • C7 Same exclusion criteria of Groups I, II, 111, W, V, VI except BMI over 25 is included.
  • N 140)
  • FIG. 2 The Effects of PhencalTM on Weight Loss. This figure shows the comparison weight in both the PHENCALTM and non- PHENCALTM groups after a two year period. At the end of the two year study, subjects taking PHENCALTM
  • FIG. 4 Additive effect of an increasing numbers of variant additive genes on the ADHD score. It showed a progressive increasing trend from 1.0 for those with only 4 or 5 variant genes, to 25.0 for those carrying 15 variant genes.
  • the p value for linear chi square test of a progressive increase in the ADHD score was ⁇ 10 .
  • FIG. 5 Additive and subtractive effect of all 29 genes on the ADHD score, and additive effect of only the additive genes.
  • the open squares on the bottom represent the r values for each gene assigned random scores that matched the frequency of the observed scores.
  • the progressively additive effect of the r ⁇ values is shown by empty squares and the additive effect of using only the positive correlations are shown by squares containing an x.
  • the lines whose points are marked by an "x" are additive genes
  • the line whose points are marked by a solid dot are additive and subtractive genes.
  • the final r " using both the additive and subtractive genes was .0001.
  • the final r using only the additive random genes was .0004. Neither was significant.
  • the commutative r was as high as .008 at the random PENK gene, this fell back to .0004 when the last random additive gene (CD8A) was added.
  • the solid dots represent the group I data, the solid squares represent the group two II data.
  • FIG. 7 Distribution of the number of variant genes for subjects with no DSM-IV ADHD symptoms versus those fulfilling DSM-IV criteria for ADHD.
  • the inventors believe that various psychological disorders are linked by a common biological substrate, a "hard-wired" system in the brain that provides pleasure in the process of rewarding certain behavior.
  • the inventors propose in this invention that an inborn chemical imbalance that alters the intercellular signaling in the nucleus accumbens or other limbic reward regions could supplant an individual's feeling of well-being with anxiety, anger or a craving for a substance ⁇ i.e. alcohol) that can alleviate the negative emotions.
  • This chemical imbalance manifests itself as one or more behavioral disorders for which the term "Reward Deficiency Syndrome" has been coined (Blum et al, 1996a).
  • the present invention identified the correlation between the predisposition to RDS and alleles of a number of genes including but not limited to the dopaminergic genes DRDl, DRD2, DRD3, DRD4, DRD5, dopamine transporter gene (DATl); Serotonin genes HTT, HTRA, HTRDb, HTRA, HTRC, tryptophan 2.3- hydroxalase (TD02); Norepinephrine genes, D ⁇ H, ADRAA, ADRAC, NT, Catecholamine metabolizing genes, MAOA, COMT; GAGA genes, GABRAA, GABRAB; Canabinoid receptor gene, CNR; Nicotinic cholinergic, CHRNA; NMD A receptor gene, NMDAR; Enkephalin genes, PENK; Androgen receptor gene, AR; Interferon gamma gene, INFG; CDA:
  • RDS and related behaviors being very complex, and the importance of a number of environmental factors, negates the possibility that one particular gene or environmental factor indeed contributes 100% as the determinant. While it is believed that, in general, the "reward cascade model", when impaired, leads to RDS behaviors, the inventors are careful to point out that while more than one gene may be responsible for a percent of the overall variance in one RDS subtrait, it may have little or nothing to do with another related RDS behavior.
  • RDS behaviors including alcoholism, SUD, smoking, BMI or obesity, pathological gambling, carbohydrate bingeing, axis 1 1 diagnosis, SAB, ADD/ADHD, CD, TS, family history of SUD, and Obesity
  • One important embodiment of this application involves a method to detect a number of genetic variants, alone or in combination, based on their individual contribution to the RDS behavior being diagnosed.
  • the inventors believe that by utilizing a combination of genes and detecting said specific polymo ⁇ hism or actual mutation one would be able to identify individuals at risk in a manner whereby greater accuracy would be achieved than if only one gene was detected as the original issued patents suggested with the DNA based detection of the dopamine D2 receptor gene. In order to provide a clearer overview of the magnitude of this potential some of the genes suspected as being involved in the reward pathway.
  • MULTI- PLEX GENESCANTM MULTI- PLEX GENESCANTM to detect a number of already associated genes in impulsive- compulsive-addictive behaviors the inventors characterized as comprising Reward Deficiency Syndrome, as well as other polygenic traits.
  • MUA Multiple Additive Associations
  • Steps to the Multiple Additive Associations (MAA) Technique The specific examples demonstrate the use of the MAA Technique to construct diagnostic assays for RDS related disorders. However, the inventors give a number of examples that are independent of psychiatric disorders and illustrate that the MAA technique can be generalized to all polygenic disorders and all polygenic traits. Thus, the inventors contemplate that the MAA technique as a procedure for all polygenic disorders. Polygenic disorders are characterized as being due to the additive effect of many genes each of which individually account for only a small percent of the variance of the phenotype. They are present to varying degrees in all individuals. Polygenic disorders are much more common than single gene disorders, affecting from 1 to 20 percent of the population.
  • Some examples are hypertension, obesity, most psychiatric disorders, multiple sclerosis, lupus erythematosis, osteoporosis, coronary artery disease, rheumatoid arthritis, osteoarthritis, weight, height, blood pressure, age (longevity), psychological traits and any other trait that is determined in part by more than one gene or allele.
  • the following teaches how the MAA technique is performed.
  • the present MAA technique has the following unique added features. First, it dramatically expands the number of genes that can be examined to thousands. It places all quantitative or dichotomous traits on the same scale by using the correlation coefficient (r), and the percent of the variance (r2) instead of the trait itself.
  • the MAA technique By examining the additive effect of multiple genes rather than examining genes one-gene-at-a-time, the MAA technique has much more power to identify the genes involved in polygenic disorders than procedures such as lod score, sib pair, haplotype relative risk (Falk and Rubinstein, 1987), and transmission disequilibrium tests (Shman and Ewens, 1996) that examine genes singly.
  • the MAA technique shows that p values for associations studies examining one-gene-at-a-time have little relevance to whether a gene is involved in a polygenic disorder or trait.
  • the MAA technique can be described as having the following steps, though certain steps are unique to the technique.
  • the first step is to identify the polygenic disorder or trait to be studied, i.e. attention deficit hyperactivity disorder (ADHD), depression, cholesterol level, weight, obesity, longevity, blood pressure, multiple sclerosis, or any other disorder or trait that is polygenic, or suspected of being polygenic.
  • ADHD attention deficit hyperactivity disorder
  • SCID SCID
  • the second step is setting a scale that measures the severity of the polygenic disorder.
  • This can be a quantitative trait or dichotomous variable (QT or DV).
  • QT or DV dichotomous variable
  • the quantitative scale could be diastolic blood pressure
  • height is studied the height in inches or cm could be used
  • obesity the scale could be weight or BMI
  • depression is studied the scale could be the number of positive DSM-IV criteria for depression, etc.
  • Scores for genes refer to assigning a 'score' of 0, 1 or 2 to the genotypes depending upon which genotypes were associated with the least, intermediate or greatest phenotypic effect.
  • the third step is to identify the candidate genes to be tested.
  • this application shows the 29 candidate genes chosen for ADHD, oppositional defiant disorder, conduct disorder, learning disorders, alcohol were those genes that play a role in the regulation of neurotransmitters including dopamine, serotonin, norepinephrine, GABA, and others.
  • any gene may potentially contribute to a polygenic trait. Selection of a candidate gene or set of genes to be used in the MAA technique would be facilitated by first selecting genes that would have some metabolic or physiological relationship to the trait being examined.
  • Genbank a computerized database
  • One of skill in the art would recognize that there are other sources of genetic information, including personal knowledge or knowledge that is not published or publicly available, which can be used to identify candidate genes for most polygenic traits and disorders, and such resources may be used in the practice ofthe MAA method.
  • candidate genes involved in a polygenic trait such as height, or susceptibility to osteoporosis
  • genes involved in bone and/or connective tissue formation, growth, and/or regulation Another trait that could be evaluated would be obesity, and candidate gene would include these genes plus the OB gene, the OB receptor gene, the neuropeptide Y gene and the neuropeptide Y receptor genes would be candidate genes.
  • candidate genes would be those relating the norepinephrine, epinephrine, steroid, and rennin metabolism.
  • genes were selected that were suspected of contributing to a polygenic trait. Of particular interest were genes suspected of contributing to RDS behaviors.
  • the criteria for the selection of a gene included indications of their involvement, from the literature, in one or more RDS behaviors or other polygenic traits of interest, and/or experimental data by the inventors, which is described below.
  • the inventors contemplate that the association of a gene or polymo ⁇ hism with a given trait may be used in a diagnostic assay for that trait, as well as provide guidance to the suitability of a gene and/or its specific polymo ⁇ hisms for inclusion in a MAA diagnostic assay for the polygenic trait of interest.
  • the traits do not have to be associated with RDS for the gene or polymo ⁇ hism.
  • DRDl Gene This particular gene in preliminary studies by the inventors did not associate with severe alcoholic probands compared to controls, thus, these additional findings with a larger sample was su ⁇ rising.
  • the DI receptor gene polymo ⁇ hism may be more associated with polysubstance abusers rather than severe alcoholics.
  • Dopaminergic Genes, Violence and Schizoid/Avoidant Behaviors Other findings support the concept of polygenic inheritance in complex personality disorders such as "pathological violence” and schizoid/avoidant behaviors (SAB).
  • SAB schizoid/avoidant behaviors
  • the inventors found a strong association between the DRD2 Al allele and "pathological violence" in adolescent probands and also found a similar association for the 10 allele of the dopamine transporter gene (DATl). Strong association was found for the DRD2A1 allele with SAB, no association was found for the D ⁇ H gene, however, albeit weaker an association was found for the DATl gene with SAB (Blum et al, 1997).
  • D ⁇ H alleles may be the most precise approach to examining the potential role of D ⁇ H in human behavioral disorders. This is the first study supporting a strong association between the DRD27 ⁇ 7L47 allele with SAB and PV. Where these complex traits do not show simple Mendelian patterns of inheritance one would not expect simple genetic answers caused by a single gene. The relationship of dopamine genes to pathological violence is further described in Specific Example 17.
  • Dopamine D2 Receptor Gene Putative mutations located in 3' and 5' non- coding regions of the gene that were maintained in linkage disequilibrium with the polymo ⁇ hic markers, may influence DRD2 transcription and/or mRNA stability and thus affect DRD2 receptor number, based on the following: a stronger association found with markers located on the flanking regions rather than at exonal point mutations; the reported lack of mutations in exons (except exon 8) of "Al -marked " DRD2 alleles; and the decreased DRD2 Bmax reported with presence of the Al marker in the absence of Kd changes (Noble et al, 1991). This may help explain the lack of association with one polymo ⁇ hism, the Taql D2.
  • PTT Positron emission tomography
  • Al + group The mean relative glucose metabolic rate (GMR) was significantly lower in the Al than the Al " group in many brain regions, including the putamen, nucleus accumbens, frontal and temporal gyri and medial prefrontal, occipito-temporal and orbital cortices.
  • GMR mean relative glucose metabolic rate
  • Decreased relative GMR in the Al + group was also found in Broca's area, anterior insula, hippocampus, and substantia nigra. A few brain areas, however, showed increased relative GMR in the Al + group.
  • DRD2 gene has been demonstrated in studies of ours (Comings et al, 1996b) and others (Noble et al, 1994; Lerman et al,
  • Dopamine- ⁇ -Hydroxylase Since D ⁇ H is located in the sympathetic nerve terminal and released into the circulation during the release of norepinephrine, the genes involved in its control could reside at loci other than the D ⁇ H gene itself. Thus, association studies between genetic markers at the D ⁇ H locus and ADHD, CD, alcoholism as well as other RDS related behaviors could be negative. On the other hand, if the serum levels of D ⁇ H are cofounded by a range of environmental factors, association studies with genetic markers of D ⁇ H could provide a more accurate assessment ofthe role ofthe D ⁇ H gene in these disorders than blood levels.
  • D ⁇ H activity results in the excessive production of dopamine which is associated with hyperactivity, aggression, self-stimulation and stereotypic movements (Randrup and Scheel-Kruger, 1996).
  • Increased frequency of the diagnosis of CD in emotionally disturbed boys with low plasma D ⁇ H levels has been reported (O'Connell et al, 1992; Rogeness et al, 1984; Rogeness, et al, 1986; Rogeness, et al, 1988; Rogeness, et al, 1987).
  • Another aspect of the invention involves association studies with the dopamine-beta hydroxylase gene and shows the first association between the D ⁇ H dinucleotide repeat polymo ⁇ hism and drug abuse patterns (Comings et al, 1996a).
  • the dinucleotide repeat polymo ⁇ hism for D ⁇ H was found to have a bimodel allelic distribution at below or above 175bp.
  • Subjects were genotyped as homozygous for low ( ⁇ 174bp) or high ( ⁇ 176bp), or heterozygous.
  • patients with the high bp homozygotes were found on the ASI to have greater number of previous drug treatments, longer history of cocaine use, more frequent IV injection of amphetamines, more frequent IV drug use. These subjects reported paternal alcoholism more often and a history of having been sexually abused in childhood.
  • For the patients with the high bp genotype was associated with lower scores on Self- Acceptance, Enlighted Second Nature, and Self-Directiveness.
  • the D ⁇ H BI allele was most associated with the variables ADHD, obsessive-compulsive, manic, oppositional defiant, and sleep. Differences included the greater association of the DRD2 Al allele with the schizoid, sexual, conduct and stuttering variables, while the D ⁇ H BI allele was more strongly associated with learning, reading and school problems. The tendency for the variables associated with school performance, such as reading, learning and grade school, to rank high in the D ⁇ H studies is especially striking in contrast to the DRD2 studies where they tended to rank at the bottom. This may be related to the role of D ⁇ H in memory.
  • Tourette's syndrome may be one of the most complex recognizable forms of RDS. Since all of the behaviors associated with the inhibition of D ⁇ H activity are common in patients with Tourette's syndrome (TS) (Comings and Comings, 1984; Comings and Comings, 1987b; Knell and Comings, 1993; Comings, 1990) an association between the D ⁇ H Taq B polymo ⁇ hism (d'Amato et al, 1989) and TS, conduct disorder, attention deficit hyperactivity disorder (ADHD), autism, or related behaviors may exist.
  • TS Tourette's syndrome
  • Al allele indicates increased risk not only for obesity, but also for other related addictive behaviors and that a BMI over 25 by itself (without characterization of macroselection [carbohydrate bingeing] or comorbid SUD) is not a sufficient criterion for association with the DRD2 Al allele (Blum et al, 1996).
  • the Cannabinoid Receptor Gene The likelihood of intravenous drug (IV) drug use may be affected by both genetic and environmental factors. To explore these gene-culture etiologic factors, 77 male non-Hispanic Caucasian substance abusers and 70 ethnically matched controls were examined. Patients were administered the Addiction Severity Index; all probands were administered the Family Environment Scale and a childhood-experiences survey questionnaire, and were genotyped for dopaminergic, cannabinoid, and GABAergic genes. The inventors found a higher prevalence of IV drug use among subjects whose genotypes included only high molecular-weight alleles of the CR/ (cannabinoid) receptor gene and low molecular weight alleles of the GABRB3 gene.
  • the CR7 gene is a tri-nucleotide repeat with at least 9 alleles, and the inventors found evidence that its effects on the phenotype may not be linearly predictable from the relative weight of the alleles, but are rather more complex in their interactions with IV drug use.
  • Electrophysiological Abnormalities and Substance Use Disorder (SUD):
  • the inventors present evidence for a significant association between decreased frontal lobe P300 amplitude and homozygosity for the >5 repeat alleles of the cannabinoid receptor gene (CBl).
  • CBl cannabinoid receptor gene
  • the present invention identifies a significant relationship between brain electrical activity mapping (BEAM) abnormalities, and association with DRD2 genotypes.
  • BEAM brain electrical activity mapping
  • the inventors believe this has commercial value as a important confirmation test for diagnosing genetically induced predisposition to RDS behaviors. It is suggested the method involves the detection of said human dopamine receptor gene Al allele and the cannabinoid receptor gene (CNRl) to accompany a standard brain map (i.e. Nicolett(TM)).
  • Serotonin Genes Defects in serotonin metabolism, and abnormalities in both blood serotonin and tryptophan levels, have been reported in many psychiatric disorders. Tryptophan 2,3-dioxygenase (TD02) is the rate limiting enzyme for the breakdown of tryptophan to N-formyl kenurenine. The inventors sought to determine if genetic variants at the serotonin HTRIA gene were associated with the phenotypic expression of TS or any of its associated comorbid behaviors. There was a significant association between the presence of the less common (shorter and the longer alleles) and scores for ADHD, CD, and oppositional defiant disorder (ODD), tics, sexual and other behaviors.
  • TD02 Tryptophan 2,3-dioxygenase
  • the T/C polymo ⁇ hism in the 5HT-2 receptor gene was also examined for possible association using Axis II Personality Disorders Structural Interview and the Addiction Severity Index (ASI) and the Buss-Durkey Hostility Scale (BHDS).
  • ASI Addiction Severity Index
  • BHDS Buss-Durkey Hostility Scale
  • the 22 genotype was associated with diagnosis of borderline personality disorder (p ⁇ 0.05) and depression (p ⁇ 0.05).
  • this marker was associated with amount of money spent on drugs (p ⁇ 0.05) and a history of rape (p ⁇ 0.0.05) and shoplifting/vandalism(p ⁇ 0.05).
  • the 22 genotype was associated with elevated scores on the Assault (p ⁇ 0.01) and Indirect Hostility (p ⁇ 0.05), subscales.
  • the 5HT-2R gene was associated with Indirect Hostility (p ⁇ 0.05), Negativism (p ⁇ 0.05), Verbal Hostility (p ⁇ 0.005), and Feelings of Guilt (p ⁇ 0.05), as well as total Hostility score (p ⁇ 0.01), but the polarity of the association was reversed (e.g., the 11 genotype was associated with higher values on all scores).
  • the gender-reversal of genotype associations suggests this is a complex gene that may interact with sex steroids.
  • Estrogen receptor the Aromatase locus, and the Arginine Vasopressin
  • Senno et al, 1992 at this gene might also be relevant to conduct disorder.
  • Two other relevant genes would be those at the aromatase (CYP 19) locus (Polymeropoulos et al, 1991 ) and the arginine vasopressin (AVP) gene (Summar, 1992).
  • Nicotinic Receptors Genes. Nicotinic receptors in the prefrontal cortex are involved in delayed response tasks, while muscarinic receptors are more involved in general working memory (Granon et al, 1995). Many studies have shown an intimate interaction between nicotine and dopamine. As with other addicting drugs nicotine produces an increase in the release of dopamine in the mesolimbic and nucleus accumbens neurons (DiChiara and Imperato, 1988; Corrigall et al, 1994; Pontiefi et al, 1996) and robust self administration (Corrigall and Coen, 1989; Corrigall and Coen, 1991 ). However, tolerance rapidly develops with repeated administration (Lapin et al, 1989).
  • NOS Neuronal Nitric Oxide Synthase
  • the nitric oxide synthase gene has recently been implicated in aggressive behavior in mice.
  • Studies of ob/ob mice show increased levels of nitric oxide synthase (NOS) compared to non-ob/ob litter mates.
  • Studies of NOS knockout mice have emphasized the important role of nitric oxide in aggressive and sexual behavior.
  • Ob/ob mice also show significantly increased levels of norepinephrine in paraventricular nucleus and lateral hypothalamus and significantly decreased levels of dopamine in the arcuate-infundibulum (Oltman, 1983).
  • nNOSla neuronal nitric oxide synthase gene
  • This genotype was also associated with having fewer friends (p ⁇ 0.04), having less friendship with the friends they have (p ⁇ 0.0005), having been married more times (p ⁇ 0.05).
  • this genotype was associated with increased Impulsiveness (p ⁇ 0.01), and decreased scores on Attachment (p ⁇ 0.05), Dependence (p ⁇ 0.02), Reward Dependence (p ⁇ 0.05), Pu ⁇ osefulness (p ⁇ 0.01), Self-Directiveness (p ⁇ 0.05), Empathy (p ⁇ 0.05), Helpfulness (p ⁇ 0.02), Pure-Hearted Conscience (p ⁇ 0.02), and Cooperativeness (p ⁇ 0.05).
  • MAO Monoamine Oxidase Gene
  • glucokinase regulatory protein GCR
  • pro-opiomelacortin CNKR
  • GNKR glucokinase regulatory protein
  • POMC pro-opiomelacortin
  • N potential mechanism related to the POMC gene is that it is the precursor for adrenocorticotropic hormone (ACTH), which acts on the cortex of the adrenal glands leading to the production of glucocorticoid.
  • ACTH adrenocorticotropic hormone
  • the POMC gene also acts as a precursor to the opioid peptides.
  • UCPl uncoupling protein
  • the present invention couples polygenic analysis of all these obesity genes, which have a number of different physiological mechanisms. These differences may allow for additive effects rather than synergism leading to a more accurate DNA based prediagnostic test. Combining the DRD2, OB, Chromosomal 2, UCP-2 and APO-D genes in one sample is the preferred embodiment rather than any gene alone. Additionally, the invention determines probands that are morbidly obese as determined by not BMI (which is not the best obesity determinant) but by percent body fat: 34% for females and 28% for males.
  • DRDl 1 1 genotype ICO Intellectual and Cultural Orientation from the FES
  • GABRB3 ⁇ 188 bp alleles 7 ARO Active Raceal Orientation from the FES
  • the fourth step is to identify one or more polymo ⁇ hisms associated with each gene.
  • These can be single base pair restriction fragment length polymo ⁇ hisms (RFLPs), or dinucleotide, trinucleotide, or other repeat polymo ⁇ hisms, such as well as variable tandem repeats, or any other marker of a gene locus.
  • RFLPs restriction fragment length polymo ⁇ hisms
  • Such polymo ⁇ hisms and methods of detection may be readily available in previously published or unpublished bodies of work, as previously described above for identifying candidate genes, in addition to the polymo ⁇ hisms disclosed herein.
  • a gene is suspected of contributing to a polygenic trait of interest, but no polymo ⁇ hism is currently available for use in the MAA technique after a review of the literature and genetic databases, one may perform genetic assays to determine polymo ⁇ hisms in a gene that may be used in the MAA technique.
  • Such assays are commonly used and described in the literature', in addition to the techniques described herein. Methods for genetic screening to accurately detect mutations in genomic DNA, cDNA or RNA samples may be employed, depending on the specific situation.
  • the present invention concerns the detection, diagnosis, prognosis and treatment of RDS diseases, and the detection, diagnosis, and prognosis of polygenic traits using the MAA technique.
  • Markers of alleles that contribute additively or subtractively to a polygentic trait, in the form of nucleic acid sequences isolated from an individual, and methods of identifying and detecting new markers to be used in MAA assays, are disclosed. These markers are indicators of a polygenic trait being assayed, and are diagnostic of the potential for an individual to exhibit a particular trait.
  • nucleic acid sequences disclosed herein may be used in the MAN technique, and thus will find utility in a variety of applications in the detection, diagnosis, prognosis and treatment or RDS or other polygenic traits.
  • applications within the scope of the present invention comprise amplification of one or more markers of a polygenic trait, using specific primers, detection of markers of a polygenic trait, such by hybridization with oligonucleotide or nucleic acid probes, inco ⁇ oration of isolated nucleic acids into vectors, and expression of RNA from the vectors.
  • the requirement to test for multiple genes in behavioral disorders and other polygenic traits is feasible and requires no new technology.
  • the polymo ⁇ hisms and variants involved are to two types, 1) single base pair changes producing restriction fragment length polymo ⁇ hisms (RFLPs), and 2) short tandem repeat polymo ⁇ hisms (STRs) [especially di-and trinucleotide repeats].
  • RFLPs restriction fragment length polymo ⁇ hisms
  • STRs short tandem repeat polymo ⁇ hisms
  • RFLP's Applied Biosystems, a division of Perkin-Elmer Co ⁇ oration, has developed a new technology and instrumentation that allows the rapid testing for PCRTM based single pair RFLP type genetic polymo ⁇ hisms.
  • This instrument, Applied Biosystems Prism 7200 sequence Detection System (TaqMan) allows for multiple gene testing.
  • This approach uses standard primers to electrophorese the section of DNA containing the restriction endonuclease polymo ⁇ hism site.
  • the unique aspect of this technology is that two short oligmers are then designed, one exactly matching one of the alleles, the other matching the other allele.
  • a fluorescent dye is attached to one end of each, and a quenching dye is attached to the other end.
  • the match is perfect, when the DNA polymerase reaches the hybridized oligmer, it is digested into nucleotides as the polymerase passes. This releasers the quencher and the dye now fluoresces maximally. However, if the oligmer does not match, instead of the nuclease digestion, the oligomer is pushed off the site and the quenching persists. Dual wavelength reading of the plate allows distinction between 11,12,22 genotypes. The entire process of reading the results on 96 samples requires less than fifteen min and the results are fed into a computer for analysis and storage. This technology, aided by a computerized workstation to set up to PCRTM reactions, allows hundreds of different RFLPs to be examined in one day.
  • PCRTM reactions for the STRs.
  • the difference is that for the STRs the primers themselves are labeled with different fluorescent dyes.
  • the accuracy necessary to identify alleles differing by only two bp is obtained from the Applied Biosystems 373 DNA sequencer which allows the sample labeled with a second dye. Each is detected by laser scanning at a different wavelength.
  • one PCRTM primer is labeled with fluorescent HEX Amidite (Applied Biosystems, Foster City, CA) or other fluorescent dye.
  • Two ⁇ l of the 10 fold diluted PCRTM product is then added to 2.5 ⁇ l deionized formamide and 0.5 ⁇ l of ROX 500 standard, denatured for 2 min at 92 C and loaded on 6% denaturing polyacrylamide gel in an AB 373 DNA sequencer.
  • the gel is electrophoresed for 5 h at a constant 25W.
  • the gel is laser scanned and analyzed using the internal ROX 500 standards present in each lane. The peaks are recognized by Genotyper( version 1.1) based on the color fragments sized by base pair length.
  • DGGE denaturing gradient gel electrophoresis
  • restriction enzyme polymo ⁇ hism analysis restriction enzyme polymo ⁇ hism analysis
  • chemical and enzymatic cleavage methods and others.
  • SSCP single-strand conformation polymo ⁇ hism analysis
  • mismatch is defined as a region of one or more unpaired or mispaired nucleotides in a double-stranded RNA/RNA, RNA/DNA or DNA/DNA molecule. This definition thus includes mismatches due to insertion deletion mutations, as well as single and multiple base point mutations.
  • U.S. Patent No. 4,946,773 describes an RNase A mismatch cleavage assay that involves annealing single-stranded DNA or RNA test samples to an RNA probe, and subsequent treatment of the nucleic acid duplexes with RNase A. After the RNase cleavage reaction, the RNase is inactivated by proteolytic digestion and organic extraction, and the cleavage products are denatured by heating and analyzed by electrophoresis on denaturing polyacrylamide gels. For the detection of mismatches, the single-stranded products of the RNase A treatment, electrophoretically separated according to size, are compared to similarly treated control duplexes. Samples containing smaller fragments (cleavage products) not seen in the control duplex are scored as positive.
  • RNase mismatch cleavage assays including those performed according to U.S. Patent No. 4,946,773, require the use of radiolabeled RNA probes.
  • Myers and Maniatis in U.S. Patent No. 4,946,773 describe the detection of base pair mismatches using RNase A.
  • Other investigators have described the use of an E. coli enzyme, RNase I, in mismatch assays. Because it has broader cleavage specificity than RNase A, RNase I would be a desirable enzyme to employ in the detection of base pair mismatches if components can be found to decrease the extent of non-specific cleavage and increase the frequency of cleavage of mismatches.
  • the use of RNase I for mismatch detection is described in literature from Promega Biotech. Promega markets a kit containing RNase I that is shown in their literature to cleave three out of four known mismatches, provided the enzyme level is sufficiently high.
  • the RNase protection assay was first used to detect and map the ends of specific mRNA targets in solution.
  • the assay relies on being able to easily generate high specific activity radiolabeled RNA probes complementary to the mRNA of interest by in vitro transcription.
  • the templates for in vitro transcription were recombinant plasmids containing bacteriophage promoters.
  • the probes are mixed with total cellular RNA samples to permit hybridization to their complementary targets, then the mixture is treated with RNase to degrade excess unhybridized probe.
  • the RNase used is specific for single-stranded RNA, so that hybridized double-stranded probe is protected from degradation. After inactivation and removal of the RNase, the protected probe (which is proportional in amount to the amount of target mRNA that was present) is recovered and analyzed on a polyacrylamide gel.
  • the RNase Protection assay was adapted for detection of single base mutations.
  • radiolabeled RNA probes transcribed in vitro from wild-type sequences are hybridized to complementary target regions derived from test samples.
  • the test target generally comprises DNA (either genomic DNA or DNA amplified by cloning in plasmids or by PCRTM), although RNA targets (endogenous mRNA) have occasionally been used. If single nucleotide (or greater) sequence differences occur between the hybridized probe and target, the resulting disruption in Watson-Crick hydrogen bonding at that position ("mismatch”) can be recognized and cleaved in some cases by single-strand specific ribonuclease.
  • RNase A has been used almost exclusively for cleavage of single-base mismatches, although RNase I has recently been shown as useful also for mismatch cleavage.
  • MutS protein and other DNA-repair enzymes for detection of single-base mismatches. Additional methods for detection of nucleic acids, and mutations are described herein.
  • nucleic Acids As described herein, an aspect of the present disclosure is 29 previously known genes whose allelic polymo ⁇ hisms are markers of polygenic traits, including markers for such polygenic traits as ADHD, oppositional defiant disorder, conduct disorder, learning disorders, alcohol, cholesterol, and LDL.
  • the nucleic acid sequences disclosed herein will find utility as hybridization probes or amplification primers. These nucleic acids may be used, for example, in diagnostic evaluation of tissue samples or employed to clone full length cDNAs or genomic clones corresponding thereto. In certain embodiments, these probes and primers consist of oligonucleotide fragments. Such fragments should be of sufficient length to provide specific hybridization to a RNA or DNA tissue sample. The sequences typically will be 10-20 nucleotides, but may be longer. Longer sequences, e.g., 40, 50, 100, 500 and even up to full length, are preferred for certain embodiments.
  • Nucleic acid molecules having contiguous stretches of about 10, 15, 17, 20, 30, 40, 50, 60, 75 or 100 or 500 nucleotides from a sequence selected from any gene that may be used in the diagnostic or treatment methods disclosed herein are contemplated. Molecules that are complementary to the above mentioned sequences and that bind to these sequences under high stringency conditions also are contemplated. These probes will be useful in a variety of hybridization embodiments, such as Southern and Northern blotting. In some cases, it is contemplated that probes may be used that hybridize to multiple target sequences without compromising their ability to effectively diagnose a polygenic trait.
  • probes and primers can be designed around the disclosed nucleotide sequences, or the sequences surrounding a polymo ⁇ hism useful as a marker, be it a gene disclosed herein or a gene latter added the set of 29 genes described herein. It is contemplated that other genes may be used to create new sets for examination of different polygenic traits, and the use of any other genes, or preferably gene polymo ⁇ hisms, in the MAA technique is encompassed as part of the invention.
  • Primers may be of any length but, typically, are 10-20 bases in length.
  • n is an integer from 1 to the last number of the sequence and y is the length of the primer minus one (9 to 19), where n + y does not exceed the last number ofthe sequence.
  • the probes correspond to bases 1 to 10, 2 to 11, 3 to 12 ... and so on.
  • the probes correspond to bases 1 to 15, 2 to 16, 3 to 17 ... and so on.
  • the probes correspond to bases 1 to 20, 2 to 21, 3 to 22 ... and so on.
  • multiple probes may be used for hybridization to a single sample.
  • the use of a hybridization probe of between 14 and 100 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 20 bases in length are generally preferred, in order to increase stability and selectivity ofthe hybrid, and thereby improve the quality and degree of particular hybrid molecules obtained.
  • Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.
  • nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of genes or RNAs or to provide primers for amplification of DNA or RNA from tissues.
  • one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence.
  • relatively stringent conditions For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50°C to about 70°C.
  • relatively low salt and/or high temperature conditions such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50°C to about 70°C.
  • Such high stringency conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating specific genes or detecting specific mRNA transcripts. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.
  • hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl 2 , 10 mM dithiothreitol, at temperatures between approximately 20°C to about 37°C.
  • Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 ⁇ M MgCl 2 , at temperatures ranging from approximately 40°C to about 72°C.
  • nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization.
  • appropriate indicator means include fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of being detected.
  • enzyme tags colorimetric indicator substrates are known which can be employed to provide a detection means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
  • the hybridization probes described herein will be useful both as reagents in solution hybridization, as in PCRTM, for detection of expression of corresponding genes, as well as in embodiments employing a solid phase.
  • the test D ⁇ A (or R ⁇ A) is adsorbed or otherwise affixed to a selected matrix or surface.
  • This fixed, single-stranded nucleic acid is then subjected to hybridization with selected probes under desired conditions.
  • the selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.).
  • hybridization is detected, or even quantified, by means ofthe label.
  • Nucleic acid used as a template for amplification is isolated from cells contained in the biological sample, according to standard methodologies (Sambrook et al, 1989).
  • the nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to convert the RNA to a complementary DNA.
  • the RNA is whole cell RNA and is used directly as the template for amplification. Pairs of primers that selectively hybridize to nucleic acids corresponding to genes of a polygenic trait are contacted with the isolated nucleic acid under conditions that permit selective hybridization.
  • primer is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process.
  • primers are oligonucleotides from ten to twenty or thirty base pairs in length, but longer sequences can be employed.
  • Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is preferred.
  • the nucleic acid:primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles,” are conducted until a sufficient amount of amplification product is produced.
  • the amplification product is detected.
  • the detection may be performed by visual means.
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of inco ⁇ orated radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax technology).
  • PCRTM polymerase chain reaction
  • PCRTM two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence.
  • An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If the marker sequence is present in a sample, the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides.
  • the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.
  • a reverse transcriptase PCRTM amplification procedure may be performed in order to quantify the amount of mRNA amplified.
  • Methods of reverse transcribing RNA into cDN A are well known and described in Sambrook et al, 1989.
  • Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641, filed December 21, 1990, inco ⁇ orated herein by reference. Polymerase chain reaction methodologies are well known in the art.
  • LCR ligase chain reaction
  • Qbeta Replicase described in PCT Application No. PCT/US87/00880, inco ⁇ orated herein by reference, may also be used as still another amplification method in the present invention.
  • a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence that can then be detected.
  • SDA Strand Displacement Amplification
  • RCR Repair Chain Reaction
  • CPR cyclic probe reaction
  • a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample.
  • the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion.
  • the original template is annealed to another cycling probe and the reaction is repeated.
  • primers are used in a PCRTM-like, template- and enzyme-dependent synthesis.
  • the primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).
  • a capture moiety e.g., biotin
  • a detector moiety e.g., enzyme
  • an excess of labeled probes are added to a sample.
  • the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence ofthe target sequence.
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Gingeras et al, PCT Application WO 88/10315, inco ⁇ orated herein by reference).
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR Genomerase binding amplification
  • the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
  • amplification techniques involve annealing a primer which has target specific sequences. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again.
  • the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization.
  • the double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6.
  • an RNA polymerase such as T7 or SP6.
  • the RNA's are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6.
  • the resulting products whether truncated or complete, indicate target specific sequences.
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • the ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
  • RNA-dependent DNA polymerase reverse transcriptase
  • the RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA).
  • RNase H ribonuclease H
  • the resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template.
  • This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence.
  • This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
  • Miller et al, PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA”) followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e. new templates are not produced from the resultant RNA transcripts.
  • Other amplification methods include "RACE” and "one-sided PCRTM" (Frohman, 1990, inco ⁇ orated herein by reference).
  • Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide may also be used in the amplification step of the present invention.
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al, 1989).
  • chromatographic techniques may be employed to effect separation.
  • chromatography There are many kinds of chromatography which may be used in the present invention: adso ⁇ tion, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography.
  • Amplification products must be visualized in order to confirm amplification of the marker sequences.
  • One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light.
  • the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.
  • visualization is achieved indirectly.
  • a labeled, nucleic acid probe is brought into contact with the amplified marker sequence.
  • the probe preferably is conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety.
  • detection is by Southern blotting and hybridization with a labeled probe.
  • the techniques involved in Southern blotting are well known to those of skill in the art and can be found in many standard books on molecular protocols. See Sambrook et al, 1989. Briefly, amplification products are separated by gel electrophoresis. The gel is then contacted with a membrane, such as nitrocellulose, permitting transfer of the nucleic acid and non-covalent binding. Subsequently, the membrane is incubated with a chromophore-conjugated probe that is capable of hybridizing with a target amplification product. Detection is by exposure of the membrane to x-ray film or ion-emitting detection devices.
  • kits This generally will comprise preselected primers for specific markers. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.
  • enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.
  • kits generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each marker primer pair.
  • Preferred pairs of primers for amplifying nucleic acids are selected to amplify the sequences specified in SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:l l, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28. SEQ ID NO:29. SEQ
  • kits will comprise hybridization probes specific for genes involved in polygenic traits corresponding to the sequences specified in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:l 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO:20.
  • SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25.
  • kits generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each marker hybridization probe.
  • D ⁇ A segments encoding a specific gene may be introduced into recombinant host cells and employed for expressing a specific structural or regulatory protein. Alternatively, through the application of genetic engineering techniques, subportions or derivatives of selected genes may be employed. Upstream regions containing regulatory regions such as promoter regions may be isolated and subsequently employed for expression ofthe selected gene.
  • this invention is not limited to the particular probes disclosed herein and particularly is intended to encompass at least nucleic acid sequences that are hybridizable to the disclosed sequences or are functional sequence analogs of these sequences.
  • a partial sequence may be used to identify a structurally-related gene or the full length genomic or cD ⁇ A clone from which it is derived.
  • Those of skill in the art are well aware of the methods for generating cD ⁇ A and genomic libraries which can be used as a target for the above-described probes (Sambrook et al, 1989).
  • nucleic acid segments of the present invention are inco ⁇ orated into vectors, such as plasmids, cosmids or viruses
  • these segments may be combined with other DNA sequences, such as promoters, polyadenylation signals, restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably.
  • a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • the sample finds a complementary probe on the Chip, it will bind; if it does not find a complementary strand, it will wash off the Chip (segments of DNA that have complementary bases are said themselves to be complementary: the fragments ATTTGCGC (SEQ ID NO:l) will bind, for example to a complementary fragment with the sequence TNAACGCG (SEQ ID ⁇ O:2).
  • the sequence and location of each probe is known, so the scanner can determine to which probe the sample has bound. Because the sequence of the probe on the Chip is known, the sequence of the sample DNA is also known, since its sequence will be complementary.
  • the use of gene chips does not require the copying of messenger RNA into cDNAs and can quantitatively detect 1 messenger rNAs and cDNAs.
  • Genotying by mass spectrometry is contemplated.
  • Sequenom San Diego, CA
  • MALDI-TOF matrix-assisted laser deso ⁇ tion/ionization-time-of-flight mass spectrometry
  • PCRTM amplification of the region of the polymo ⁇ hism with biotin attached to one of the primers is conducted (Jurinke et al, 1997).
  • immobilization of the amplified DNA to strepavidin beads occurs (Jurinke et al, 1997)
  • hybridization of a primer adjacent to the polymo ⁇ hism site is done (Braun, Little, K ⁇ ster, 1997).
  • extension with DNA polymerase past the polymo ⁇ hic site in the presence of dNTPs and ddNTPs which are not present in the deoxyform form is done. When suitably designed according to the sequence, this results in the addition of only a few additional bases (Braun, Little, K ⁇ ster, 1997).
  • the DNA is then processed to remove unused nucleotides and salts.
  • the short primer + polymo ⁇ hic site is removed by denaturation and transferred to silicon wafers using a piezoelectric pipette (O'Donnell et al, 1997).
  • the mass of the primer + polymo ⁇ hic site is then determined by delayed extraction MALDI-TOF mass spectrometry (Li et al, 1996; Tang et al, 1995). Single base pair and tandem repeat variations in sequence are easily determined by their mass. This final step is very rapid, requiring only 5 sec per assay. All of these steps are robotically automated. This technology has the potential of performing up to 20,000 genotypings per day.
  • This technology is rapid, extremely accurate, and adaptable to any polymo ⁇ hism. It has a significant advantage over chip technology in that it is much more accurate, can identify both single base pair and short tandem repeat polymo ⁇ hisms, and adding or removing polymo ⁇ hisms to be tested can be done in a few sec at trivial cost.
  • Drug abuse including IV drug use DI, D2, D4, CNRl, GABAB3, TD02, HT-2R,
  • SUD sub-class Stimulant Withdrawal COMT, nNosla
  • SUD sub-class Stimulant Withdrawal: Depression TD02
  • Compulsivity subclass Pathological Gambling DI, D2, TD02
  • Compulsivity subclass Sexual Compulsions
  • Obesity subclass APOD, CHROME-2
  • Obesity subclass DATl, OB
  • Attention Deficits subclass Tourette's DI, D2, TD02, D ⁇ H, MAOA(X), HTRIA
  • ADHD HTRIA ADHD HTRIA
  • D2 DATl
  • D ⁇ H ADHD HTRIA
  • TD02 Attention Deficits subclass: ADHD HTRIA, D2, DATl, D ⁇ H, TD02
  • the fifth step would be to assign a score to the genotypes based on independent studies showing which genotypes are associated with the highest quantitative scores and which are associated with the lowest scores.
  • the scoring of the genes is based on the use of ANOVA to examine the magnitude of the mean Kellgren score for each genotype of each polymo ⁇ hism. These studies are performed in a set of subjects independent of those used for the MAA.
  • DRD2 For example, for the Taql A1/A2 polymo ⁇ hism of the dopamine D 2 receptor gene, DRD2, all studies have shown that the 1 allele is associated with a range of impulsive, compulsive, additive behaviors, and some studies show that the heterozygous genotype, 12, was associated with the highest scores and the 1 1 and 22 genotypes with the lowest scores.
  • STEP 6 Set up a dummy polygenic or PG variable, by progressively adding the scores of each gene. For example, if for case 1 , the score for the first gene examined was 2, and for the second gene the score was 1, the PG score variable would be 3.
  • the correlation coefficient, r, for 1 thru n cases against the quantitative trait variable score would be determined by regression analysis, preferably using any off- the-self statistical program. The percent of the variance is r . Then, for cases 1 thru n, the gene score for the 3rd gene is added and the process repeated. The final results are plotted.
  • STEP 7. Perform univariate regression analysis of PG versus QT or DV.
  • the following is an example generalized to any quantitative trait or dichotomous variable.
  • each additional gene score is added, a regression analysis performed, the second gene score is added, the regression analysis is repeated. This is continued until all of the tested genes have been added.
  • One preferred method of analyzing the data would be to enter all the data into a statistical package such as SPSS. Using the values for the DRD2 gene as described under Step 5 above, the syntax file for the SPSS program would be opened and the following sample algorithm set up.
  • the PG variable is set to 0. Since the DRD7, DRD2, DRD3 and DRD4 genes were scored as 0 or 2 they required only a single line increasing PG by 2 if the subject carried the DRDl Ddel 1 1 genotype, and/or the DRD2 Taql 12 genotype and/or the DRD3 Mscl 1 1 or 22 genotype, and/or DRD4 46,47, or 77 genotype. By contrast the DRD5 and DATl genes were scored as 1 or 2, requiring two lines of code. Regression analysis is performed against the PG score against the QTV (ADHD score), as each gene is added.
  • ADHD score QTV
  • STEP 8 Plot the results.
  • the results are plotted with the progressively added genes on the X axis and the r2 on the Y axis, as shown in the line in FIG. 5 showing the additive and subtractive genes for ADHD.
  • STEP 9 Repeat the procedure using only the additive genes. This is shown in the line in FIG. 6 showing the additive genes.
  • Certain anti-craving substances which reduce the desire for euphoriants by virtue of enhancing the effectiveness of endogenous and or exogenous neuropeptides and neurotransmitters such as dopamine release in the nucleus accumbens, significantly reduces aberrant craving behavior, termed "RDS" behavior, for euphoriant substances to include but not limited to alcohol, cocaine, opiates, nicotine, glucose or other sugars, as well as certain acts such as sexual, gambling, aggression and violence.
  • RDS behaviors have a common genetic basis, and based on adequate supplies of neurotransmitters in the meso-limbic system.
  • the final pathway of reward involves dopaminergic activation at receptor sites (DI, D2, D3, D4, D5 and subtypes) and the density of these receptors are determined by their respective genes as well as genes responsible for dopamine, synthesis, storage, release and metabolism.
  • This invention proposes to couple the use of enhancement of synaptic dopamine release via enkephalinase inhibition and an enkephalin releaser such as kyoto ⁇ hin (Tyr-Arg) or its stable analog, TyrD-Arg, to promote a chronic occupancy of D2 receptors with potential for D2 receptor proliferation or up-regulation (Fitz et al, 1994).
  • an enkephalin releaser such as kyoto ⁇ hin (Tyr-Arg) or its stable analog, TyrD-Arg
  • This invention involves, in part, the idea of polygenic inheritance and therefore promotes the concept of multiple gene loci as determinants for identifying high risk candidates for RDS.
  • Work involving quantitative trait loci studies in genetically distinguishable mouse strains provided negative evidence for the one single gene mechanism theory. Fifteen strains of mice were tested on their reaction to alcohol, mo ⁇ hine and methamphetamine. These strains were found to react differentially to these three substances, which suggests that different genes determine the susceptibility to different addictive substances.
  • the pattern of findings further suggests that genetically influenced sensitivity to alcohol is not a monolithic phenomenon. Rather, it is specific to the particular response variable studied (Crabbe et al, 1994).
  • an aspect of this invention is the genotyping certain of the above specified genes, and other genes suspected of contributing to RDS behaviors or other polygenic traits, to obtain haplotype profiles would be an important aspect of targeted treatment of RDS, related behaviors, or other polygenic traits.
  • the inventors can utilize this knowledge to assist the clinician in pharmacogenetically targeting treatment.
  • the invention couples the use of genotyping and precursor amino-acid loading, enkephalinase inhibition, enkephalin-induced release, narcotic antagonism, and chromium piccolinate or niccotinate for the preferential enhancement of dopamine in the nucleus accumbens as anti-craving compositions.
  • the inventors propose that an alteration in any of the genes that are involved in the expression of molecules in the reward cascade (which might be as many as 100 or more) might predispose an individual to RDS and related behaviors, even including ADHD and violence. While the above may be true, it is very difficult at this time to predict which particular genes are involved, that is, until the disclosure of the present invention as it relates to the MAA technique. However evidence for a role of at least the D2 dopamine receptor gene is profound and the inventors believe that the D2 dopamine receptor gene represents a major "reward gene" important in RDS and related behaviors.
  • the preferred embodiment of this invention is to couple the TaqAl DRD2 alleles (Al , BI , Cl, haplotype polymo ⁇ hism between exon 6-7) and targeted treatment outcomes (i.e. attention processing, prevention of regained weight, smoking cessation, reduction of carbohydrate bingeing, reduction of AMA rate, elapse prevention in polysubstance dependence, and reduction in violent behavior, among others).
  • targeted treatment outcomes i.e. attention processing, prevention of regained weight, smoking cessation, reduction of carbohydrate bingeing, reduction of AMA rate, elapse prevention in polysubstance dependence, and reduction in violent behavior, among others.
  • the inventors will also provide examples of a number of likely genotypes based on specific genes as listed above which should respond best to certain drug classes.
  • hypothalamic serotonergic neurons innervate and activate met-enkephalinergic neurons that, in turn, inhibit ⁇ -aminobutyric acid (GABA) neurons, which then activate DA neurons of the ventral tegmentum.
  • GABA ⁇ -aminobutyric acid
  • These DA neurons then project to the Acb and to Cluster Al (CA1) cluster cells in the hippocampus, where the neurotransmitter DA acts as the primary reward substrates (Stein and Beluzzi, 1978).
  • CA1 Cluster Al
  • the nucleus accumbens and enkephalins in this complex circuit is important (Heidreder et al, 1988).
  • DA release in the striatum was induced after local application of enkephalinase inhibitor, which suggests regulation by delta receptor stimulation (Chesselet et al, 1981 ).
  • Kelato ⁇ han may also protect against possible cholecysterkinine-8 (CCK-8) degradation by brain peptidases.
  • This important satiety neuropeptide is colocalized with DA in the nucleus accumbens, and there is a close interaction between CCK-8, DA, and endogenous opioid peptides (Koob, 1992).
  • compositions of the present invention are especially designed to enhance these food inhibitory neurotransmitters through precursor amino acid loading, including 1-tryptophan (5-HT precursor), l-phenylalanine (DA and NE precursor), as well the enkephalinase inhibitor d-phenylalanine (Blum et al, 1986).
  • 5-HT precursor 1-tryptophan
  • DA and NE precursor l-phenylalanine
  • d-phenylalanine Blum et al, 1986.
  • the inventors are currently performing research on Lewis rats to establish the effect of these agent categories. Lewis rats seem to have proclivity for polysubstance abuse, and serve as an important animal model for RDS behavior.
  • DPA D-phenylalanine
  • NTX naltrexone
  • TA Ty-D-Arg
  • compositions for Treating RDS Although a variety of disorders are categorized as belonging to RDS and affect the dopaminergic system, one cannot treat all individuals suffering from RDS alike. Numerous other systems can be affected and require simultaneous treatment in order for an overall success to be achieved. In particular, disorders which involve drug or substance abuse must be treated individually. Thus although the overall treatment comprises giving a patient an effective dose of enkephalinase inhibitors, enkephalin releasers and amino acid precursors for the dopaminergic system, additional components are utilized to enhance the overall effect of treatment. Certain therapeutic agents are favored by the "gene-D2 receptor deficiency theory". It is well established that DRD2A1 carriers have low levels of DRD2 receptors.
  • An important embodiment of this invention relates to the providing of certain precursor amino-acids, a trace metal such as Chromium piccolinate and/or chromium niccotinate, an enkephalinase inhibitor, a narcotic antagonist and an ekephalin releasing agent in therapeutic amounts (alone or in combination) to in fact cause a natural release of dopamine to induce D2 proliferation, especially in D2A1 carriers.
  • a trace metal such as Chromium piccolinate and/or chromium niccotinate
  • an enkephalinase inhibitor e.g., a narcotic antagonist
  • an ekephalin releasing agent in therapeutic amounts (alone or in combination) to in fact cause a natural release of dopamine to induce D2 proliferation, especially in D2A1 carriers.
  • TROP AMINETM A formulation developed by the prime inventor called TROP AMINETM has been studied and utilized in patients. TROPAMINETM has been compared to other medications, and shows a major benefit in terms of both
  • Medication Patients Physicians Medication Patients Physicians lmipramine 2,885 122 2.48 Phenobarbital 100 3 NR
  • a component of this embodiment is to first genotype the patient and then based on his/her genotype provide the appropriate cocktail.
  • the inventors have developed an appropriate cocktail which is described herein.
  • Table 5 summarizes the uses of compositions of the present invention as improved with specific genotypes.
  • Tables 6-16 list the preferred components of these compositions that are useful for the treatment of various disorders. Also, see Tables 17-19 for a brief schematic of how certain elements effect reward induced by stimulants (cocaine, etc.), opiates and sedative-hypnotics.
  • AlcotrolTM Substance Use Disorder Anti-craving, reduced anxiety, DBPC- DI (Increased frequency of Dde — special emphasis on reduced-relapse, reduced against Inpatient homozygosity of the Al allele) sedative-hypnotic abuse medical advice rates (AMA), D 2 (Taq Al, BI , exon " haplotypes, (i.e. alcohol, opiates, improved physical and BESS Cl) barbiturates) scores
  • PHENCALTM Obesity (carbohydrate weight loss, reduced bingeing DBPC - D 2 ro ? I Al, Bl, Cl or exon °" bingers, anorexia, episodes, positive body Outpatient haplotype bulemia) composition changes, prevention HTR2A - C allele homozygous of lost weight regained OB - homozygosity for ⁇ 208 BP alleles of 1875 dinucleotide repeat polymo ⁇ hism human chromosome 2 microsatellite polymo ⁇ hism, D2S1788
  • PMXTM PMS reduced pain or cramps, reduced OT Outpatient Same as for AlcotrolTM and headaches, improved overall CocotrolTM mood POMC/Pre- enkephalin/Dyno ⁇ hin/O ⁇ han
  • Alternate composition is restricted to the following: D-phenylanine 500 mg per capsule 6 ⁇ day; Tye-D-Arg - 1 mg per capsule 6 ⁇ day; Naltrexone 50 mg per capsule 1 to 3 per day.
  • D-phenylanine 500 mg per capsule 6 ⁇ day Tye-D-Arg - 1 mg per capsule 6 ⁇ day
  • Naltrexone 50 mg per capsule 1 to 3 per day *GABRB3-homozygousity of dinucleotide repeat ⁇ l 85 bp HTRIA - TC polymo ⁇ hism HTR2A - C allele (homozygosity)
  • ABBREVIATIONS :
  • ADHD Attention Deficit Hyperactivity Disorder
  • DBPC double-blind, placebo-controlled
  • the inventors recommend administering the "core" Neutraceutical three times per day (before meal times). If the patient has a persistent set of addictive/impulsive/compulsive behaviors, or significantly severe addictive/impulse/compulsive behaviors, the inventors recommend administering the "Core" Neutraceutical plus the appropriate adjunctive Neutraceutical packet at the appropriate times daily.
  • the patient should take the white colored capsules at the times in which he/she is directed to take the "core" neutraceutical.
  • the patient should take the orange colored capsules before the morning meal.
  • composition per capsule in mgs
  • Vitamin B-6 P-5 phosphate 0.330
  • composition per capsule in mgs
  • Vitamin B-6 P-5 phosphate
  • Vitamin B-l 2 0.670
  • composition per capsule in mgs
  • Vitamin B-6 P-5 phosphate
  • Vitamin B-l 2 0.670
  • composition per capsule in mgs
  • Vitamin B-6 P-5 phosphate
  • composition per capsule in mgs
  • Vitamin B-6 (P-5 phosphate) 1.000
  • composition per capsule in mgs
  • Vitamin B-6 P-5 phosphate
  • Vitamin B-l 2 0.001 pharmaline 50 huberzine 150 ⁇ g
  • composition per capsule in mgs
  • Vitamin B-6 P-5 phosphate
  • composition per capsule in mgs
  • Vitamin B-6 P-5 phosphate
  • composition per capsule in mgs
  • Vitamin B-6 P-5 Phosphate
  • composition per capsule in mgs
  • Vitamin B-6 P-5 Phosphate
  • GABA A receptor antagonists Inhibition GABA ⁇ receptor agonists Facilitation Opioid receptor antagonists
  • Dopamine receptor antagonists Inhibition 6-HT receptor agonists
  • Noradrenaline synthesis inhibitors Inhibition
  • D-phenylalanine, D-leucine and any D-amino acid including hydrocinnamic acid are included in the treatment formulations.
  • the enkephalinase inhibitors are included in the formulations, including, but not limited to: certain protein synthesis inhibitors, such as bacitracin, bestatin, and puromycin; peptide amino acids such as mono free form amino acids of the D-form, di- and tripeptides of the essential amino acids in the D- form; thiol benzyl amino acids (2-[mercapto-3-phenyl-propanoyl]-L-leucine); carboxyalkyl methyl esters (N-[(R,S)-2-carbethoxy-3-phenyl propanol]-L leucine); as well as a number of other structurally unrelated compounds such as secobarbitol, pyrophosphate, O-phenanthroline, phosphamidon,
  • the inventors realized that by also supplying an enkephalin releaser they could dramatically improve the response of the patient to treatment.
  • the enkephlin releasers Tyr-Arg and Tyr-D-Arg are also included in the treatment formulations.
  • the linkage of numerous genetic alleles to a wide variety of impulsive, compulsive and addictive disorders suggest that a common mechanism is affected by both psychostimulants as well as non-psychostimulants to cause a preferential release of dopamine (DA) into the medial nucleus accumbens (Acb).
  • DA dopamine
  • compositions that alter neurotransmission of the "reward cascade” should have beneficial effects for substance and behavioral disorders.
  • Abused substances and behavioral disorders include, but are not limited to, alcohol, cocaine, nicotine, glucose, Cannabis, opiates and opiate derivatives, gambling, sexual compulsion, hyperactivity, chronic violent behavior and stress disorders, and also symptoms related to premenstrual syndrome (PMS).
  • An important component of this embodiment is to first genotype the patient and then based on his/her genotype provide the appropriate cocktail.
  • the inventors have developed an appropriate cocktail which is described herein.
  • Synthetic agonists are not preferred therapeutic agents.
  • a given agonist may act on several receptors, or similar receptors on different cells, not just on the particular receptor or cell one desires to stimulate.
  • tolerance to a drug develops (through changes in the number of receptors and their affinity for the drug), tolerance to the agonist may likewise develop.
  • a particular problem for example, bromocryptine or methamphetamine, is that it may itself create a drug dependency. It is known that both bromocryptine is self-administered by rhesus monkeys (Woolverton, et al. 1984).
  • DPA D-phenylalanine
  • releasers are effective only if they have something to release. They will not cure a state of dopamine depletion. Indeed, the inventors would be concerned that dopamine releasers by themselves would exacerbate the chronic depletion of dopamine.
  • precursors use a naturally regulated pathway. The precursor is converted to the neurotransmitter only when needed, and then the body distributes the product on the basis of need.
  • d-fenfluramine acts via decreases in daily energy intake varying between 13-25%. D-fenfluramine also works by decreasing the desire for fatty foods. The notion here is that with the PHENCALTM formula even more than what is generally accepted for the first FDA approved drug for weight reduction since d-amphetamine and other stimulants has been achieved. The inventors believe that the results on utilizing amino-acid precursor loading and enkephalinase inhibition is by far the greatest advance to prevent weight regain.
  • the basic treatment regime for RDS behaviors should contain at least one of the substances below alone or in combination (Table 20). This list contains the major constituents ofthe treatment in terms of either drug or "neutraceutical” (for specific formulas see Tables 6-16):
  • Vitamin B6 (as pyridoxine 1 to 1000 mg
  • Phrase typing of Alcoholics and Cocaine Abusers Genotyping Type A and B Probands.
  • researchers have been testing the concept of classifying or subtyping, alcoholics as Type A or Type B. and are now finding the concept useful in studying cocaine abuse.
  • Subtyping is a system for classifying and studying individuals who share one or more common characteristics.
  • Subtyping alcoholics provides a greater understanding of the complex interactions between genetic, personality, and environmental risk factors in the development of alcoholism, as well as resiliency against succumbing to these risk factors.
  • Type B alcoholism appears to have several characteristics: it is more related to hereditary factors than type A; it is more likely to occur among men; type B's are more impulsive and tend to have stronger family history of alcohol abuse; they have more childhood conduct disorder problems and more severe alcohol dependence, polydrug abuse, and psychiatric disorders, especially anti-social personality.
  • Type B scored higher than Type A in assessments of sensation seeking, aggression, criminality, violence and impairment of social adjustment.
  • the former type also used greater amounts of cocaine more frequently and for longer durations than Type A cocaine abusers.
  • Type B's also suffered more adverse effects from their drug use, such as unconsciousness, and violence (among others), and they reported a greater degree of additional drug abuse to relieve withdrawal distress.
  • Type B abusers become involved with cocaine at younger ages for: first use, first binge, first regular use, first daily use and first symptoms of addiction.
  • An aspect of the present invention combines new work utilizing molecular genetic diagnosis with previously identified markers of Type B behavior to more accurately typograph both the alcoholic and cocaine abuser.
  • the new genetic findings suprisingly fit the Type B associated variables (Ball et al, 1995).
  • the following associations with only the DRD2 gene in by itself closely associates with almost all of the Type B parameters suggested by Ball et al, 1995.
  • the evidence provided comes from a number of studies concerned with genotyping non-Hispanic Caucasians with polymo ⁇ hism of the dopamine D2 receptor gene (see Table 20-A).
  • Attention Processing Disorder One aspect of the invention is the treatment of attention processing disorder and other RDS related syndromes.
  • the inventors base this treatment on the fact that attentional processing is governed by neurotransmitter function and certain specific neurotransmitters are responsible for normal brain cognitive functioning, which could be modulated by certain precursor amino acids. Understanding of electrophysiological functioning ofthe brain resides in the biogenetic aspects of the chemical mediators known to be involved in attentional processing.
  • ADHD attention- deficit/hyperactivity disorder
  • This invention provides a composition of matter (including the use of phenylalanine) which promotes neurotransmitter manipulation which leads to natural dopamine release which overcomes PHD as well as facilitate the proliferation of dopamine D 2 receptors via occupancy by synaptic dopamine.
  • Serotonin (5-hydoxytryptamine, 5HTP) is a CNS neurotransmitter. It is also found in the enterochromaffin system of the intestine, and in blood platelets. This neurochemical is biosynthesized by first hydroxylating L-tryptophan to obtain 5 -hydroxytryptophan and then decarboxylating the latter to obtain serotonin. The hydroxylation (the rate-limiting step) is performed by the enzyme tryptophan hydroxylase, while the decarboxylation is accomplished by the ubiquitous enzyme L-aromatic acid decarboxylase. This enzyme requires pyridoxal phosphate as a cofactor.
  • Serotonin is metabolized into 5-hydroxyindole-acetic acid by monoamine oxidase. This metabolite is then excreted in the urine.
  • Central brain serotonin mechanisms may be important in the control of mood and behavior, motor activity, feeding and control of hunger, thermoregulation, sleep, certain hallucinatory states, and possibly some neuroendocrine control mechanisms in the hypothalamus.
  • Cocaine apparently reduces uptake of the serotonin precursor tryptophan, thereby reducing serotonin synthesis.
  • Cocaine also reduces tryptophan hydroxylase activity.
  • cocaine decreases serotonergic action (Reith et al, 1985).
  • tryptophan was injected into the bloodstream, the levels of tryptophan and serotonin in the brain were elevated nine- and two-fold, respectively. Infusion of tryptophan in neurological patients with both depression and insomnia resulted in six-fold elevations in cortical tryptophan levels (Gillman et al, 1981). Comparison of tryptophan (50 mg/kg) and tyrosine (100 mg/kg) or placebo in a double-blind crossover study in eight healthy men (Lieberman et al, 1983). Tryptophan, but not tyrosine. significantly reduced pain discriminibility.
  • tryptophan hydroxylase is not saturated, i.e. the enzyme is not working to full capacity and thus tryptophan hydroxylase activity is significantly affected by L-tryptophan.
  • the amount of available free L-tryptophan is dependent on a number of factors including the concentration of circulating L-tryptophan in the plasma at the rate of its uptake in the brain and presynaptic terminals.
  • the inventors contemplate using L-tryptophan or 5HTP to restore the serotonergic system disrupted by cocaine.
  • 5HTP is not as useful as a therapeutic agent.
  • the rate of entry of L-tryptophan into the brain depends upon the ratio of free-bound tryptophan in the plasma, and this ratio is influenced by the concentration in the blood of neutral amino acids, insulin and pharmaceutic agents, which compete for the plasma protein binding sites, as well as for the tryptophan-uptake sites.
  • 5HTP is taken up by neurons other than just 5HT neurons; therefore the increases in 5HT synthesis are not selectively limited to serotonin neurons.
  • Inhibitors of enzymes involved in 5HT synthesis include irreversible tryptophan hydroxylase inhibitors (DL-parachlorophenylalanine, 6-Flurotryptophan and L-propyldoracetamide) and inhibitors of 5HTP decarboxylase (carbidopa and l-methyl-5HTP).
  • Serotonin can be released into the synaptic cleft by the process of exocytosis in response to action potentials and to drugs.
  • Facilitation of 5HT release can be accomplished with cocaine, (+)-amphetamine, methamphetamine, fenfluramine, parachloramphetamine, clorimipramine (clomipramine) and amitriptyline.
  • 5HT receptor agonists include LSD, quipazine, N,N-dimethyl-tryptamine (DMT).
  • 5HT receptor antagonists include cyproheptadine, methysergide, LSD, 2-bromo-CSD (BOL), ketanserin, xylamidine, cinanserin and 1 -(-)-cocaine.
  • Inactivation of 5HT involves high-affinity energy-dependent active-transport mechanism which exists to remove 5HT from the synaptic cleft back into the presynaptic neuron.
  • Inhibitors of neuronal uptake of 5HT include the tricyclic anti-depressants
  • Enhancer/releaser of Opioid Peptides Enhancer/Releaser of Opiod Peptides
  • An aspect of this invention is the use of substances which inhibit the destruction of neuropeptidyl opiates. These opiates promote the synthesis and release of dopamine. It has been shown that the administration of opiate-like substances to animals increases the rate or striatal DA biosynthesis and metabolism, an effect which is mediated by special opiate receptors located on nigrostriatal dopaminergic terminals (Clouet et al, 1970; Biggio et al, 1978; Regiawi, 1980).
  • Cocaine also affects opiodergic action. With chronic exposure cocaine to rats, dose-dependent alteration of naloxone binding was observed. Opiate receptor density was significantly decreased in several brain structures, while it was increased in the lateral hypothalamus. It appears that opiate binding was specifically affected in "reward centers” and not in other regions (Hammer et al, 1987). Furthermore, naloxone, in another study, effectively blocked the threshold lowering action of cocaine in reward centers of the brain (Bain and Korwetsky, 1987). Moreover, cocaine appears to affect the analgesic action of certain opiates (Misra et al, 1987). The inventors believe that the reinforcing action of cocaine may be mediated in part by opiate systems in brain reward centers, which are altered by chronic cocaine exposure.
  • Narcotic drugs were found to act at various "opiate receptors.”
  • the brain and other nervous tissue were found to possess endogenous opioids (EO).
  • EO endogenous opioids
  • the related pentapeptides, methionine and leucine-enkephalin were identified in the brain (Hughes et al, 1975).
  • the enkephalins activate both delta and mu receptors, while beta endo ⁇ hin activates the epsilon receptor.
  • Endocrinologists were able to show that B-lipotropin (B-LPH), already recognized as a pituitary hormone, contained the Met-enkephalin sequence of five amino acids, and that B-LPH was hydrolyzed to an active opioid, B-endo ⁇ hin (Li et al, 1976).
  • the endo ⁇ hin family includes the large precursor, pro-opiocortin, B-LPH, and B-endo ⁇ hin.
  • the second family of EO's is the enkephalin family. Both [Met]enkephalin and [Leu]-enkephalin are derived from a large peptide precursor containing both sequences.
  • the third family are kappa agonists, such as dyno ⁇ hins 1-13 and 1-17. These CNS components antagonize mo ⁇ hine actions.
  • Dyno ⁇ hin may act as a precursor of Leu-enkephalin which forms the N-terminus; conversion to the subendo ⁇ hin form (E5) will then result in altered receptor affinity (kappa to delta), illustrating a possible new regulatory role for enzyme modulating ligand expression.
  • Peptides from each family seem to act both as neurotransmitters and as neurohormones.
  • the pentapeptide enkephalins are localized in nerve terminals and are released from neurons upon stimulation.
  • Leu- and Met-enkephalins are released from the adrenal medulla into the blood and act as neurohormones.
  • Beta-endo ⁇ hin is released from the pituitary gland into blood and it may act as a neurotransmitter in discrete areas of the brain (Bloom et al, 1978).
  • Both endo ⁇ hins and enkephalins produce biochemical and pharmacological responses, including tolerance, dependencies and abstinence, similar to those produced by narcotic analgesic drugs when the EO's are administered to man or animals.
  • the endogenous opiates like the narcotic drugs, are members of the class "opioids.”
  • Enzymes which degrade enkephalins (E5) are generally called “enkephalinases.”
  • Enzymes acting as enkephalinases include soluble and particulate bound aminopeptidases (Hersh, 1981) and others acting at the Gly3-Phe4 site such as peptidyl dipeptidases or metalloendopeptidases (Benuck et al. 1982; Schwartz et al, 1980).
  • the metalloenzyme carboxypeptidase A cleaves enkephalin leaving Tyr-Gly-Gly-C and the terminal dipeptides Met-Phe or Leu-Phe.
  • One strategy to deal with the degradation is to provide E5 surrogates.
  • tissue enzymes include: a) modification of N-terminal-Tyr inasmuch as tyrosine-modified analogs of methionine enkephalinase resist degradation (Coy and Kastin, 1980); b) presence of a D-amino acid in position 2 to block effects of amino peptidases: and/or c) modification or presence of a D-amino acid in positions 3-5 to block action of peptidyl dipeptidases or other enzymes acting at the Gly3-Phe4 bond.
  • D-Ala-enkephlamide or FK 33-824 as mu agonists
  • delta agonists such as enkephalin- Arg-Phe
  • dyno ⁇ hin 1-13 or 1-17 which are kappa agonists (Wisler et ⁇ /., 1981).
  • Gly-Gly-Phe-Met or Gly-Gly-Phe-Leu, Phe-Met, Phe-Leu can act as inhibitors of enkephalin and it is likely that larger enkephalin-type forms themselves also have inhibitory properties.
  • enkephalinase inhibitors includes but is not limited to D-Phenylalanine (DPA), DL-Phenylalanine (DLPA), hydrocinnamic acid, and D-amino acids such as D-Leucine.
  • DPA D-Phenylalanine
  • DLPA DL-Phenylalanine
  • hydrocinnamic acid hydrocinnamic acid
  • D-amino acids such as D-Leucine
  • enkephalinase inhibitors selected from a group consisting of certain protein synthesis inhibitors (bacitracin, bestatin, and puromycin), peptide amino acids (free, D-form monoamino acids, di-and tripeptides of the essential amino acids in the D-form, thiol benzyl amino acids, (e.g., 2-[mercapto-3-phenylpropanoyl]-L-Leucine), carboxyl alkyl methyl esters, N-[(R,S)-2-carbethoxy-3-[phenyl propanol]-L leucine), benzomo ⁇ han-enkaphalins, and other, structurally unrelated compounds such as secobarbital, pyrophosphate, o-phenanthroline, phosphamidon, Z-leucine-NHOFI, and Z-glycine-NHOH.
  • protein synthesis inhibitors bacitracin, bestatin, and puromycin
  • peptide amino acids free
  • Dipeptides D-Phe-D-Leu and D-Phe-D-Met and the polypeptide L-Tyr-Gly-Gly-D-Phe-D-Leu and L-Tyr-Gly-Gly-D-Phe-D-Met, together with D-Phe, D-Leu, and hydrocinnamic acid, are of particular interest.
  • D-phenylalanine has been known to inhibit carboxypeptidase A (Hartruck and Lipscomb, 1971) and more recently has been shown to possess analgesic properties (Ehrenfeld et al, 1978; Delia Bella et al, 1979) as well as antidepressant action (Beckmann et ⁇ /., 1977).
  • D-phenylalanine As an inhibitor of enkephalinases it was shown that the compound indeed significantly reduced degradation of the oligopeptides (D-Ala2-D-Leu5) enkephalin (DAPLE) and Tyr-D-Ala-Gly-Phe (TAAGP), in rat intestinal mucosa (Gail et al, 1983).
  • D-phenylalanine was much less effective when studied in vitro for inhibitory activity against both enkephalinase A and enkephalinase B activity obtained from calf brain (Amsterdam et al, 1983).
  • the addition of just one amino acid to form the dipeptide D-Phe-Tyr markedly enhances the inhibitory potency.
  • D-phenylalanine has been shown to inhibit the degradation of both enkephalins and B-endo ⁇ hin. It works better on the enzymes regulating enkephalin breakdown as compared to the enzymes regulating B-endo ⁇ hin. Its activity is also tissue-specific; in the hypothalamus, enkephalinase is 80% inhibited and endo ⁇ hinase 5%; in the cortex, enkephalinase 60%, but endo ⁇ hinase only 18%; in the striatum, enkephalinase 78% and endo ⁇ hinase 10%; and, in the spinal cord, enkephalinase 84%, endo ⁇ hinase 40% (Ehrenfeld et al, 1981).
  • Another aspect of this invention is to combine an enkephalinase inhibitor with an enkephalin releasing agent.
  • the rationale for this is that by doing so the inventors could significantly enhance the effect of enkephalin on its respective opiate receptor sites (e.g., delta or mu).
  • the inventors would prefer to use the peptide Tyr-Arg (Kyoto ⁇ hin), or its stable analog, Tyr-D-Arg, which has been shown to be analgesic and to enhance intracellular calcium in synaptosomes in rat brain striatal slices. These substances appear to be putative methionine-enkephalin releasers acting by an unknown mechanism (Ueda et al, 1986).
  • Tyr-Arg the substance known as Kyoto ⁇ hin (Tyr-Arg) may be used at a daily dosage range of 15 ⁇ g-15 mg (Takagi et al, 1979).
  • the more stable analog Tyr-D-Arg, at a daily dosage range of 15 ⁇ g-15 mg may be substituted as a enkephalin releaser (Tajima et al, 1980; Ueda et al, 1986).
  • an enkephalin releaser may be combined with an enkephalinase inhibitor to achieve a high degree of enkephalinergic activity at the synapse to further augment the release of reuronal dopamine.
  • GABA Gammabutyric Acid
  • GABA is an inhibitory neurotransmitter which controls the release of dopamine (Gessa et al, 1985). It seems to reduce seizure activity during alcohol withdrawal.
  • GABA gamma-aminobutyric acid
  • GABA gamma-aminobutyric acid
  • GABA glutamic acid decarboxylase
  • GAD is found exclusively in the cytoplasm of synaptic GABA nerve terminals.
  • the basic control of GABA synthesis is GAD which seems to be the rate limiting step in GABA synthesis.
  • GABA can influence FAD activity by end-product inhibition. Saturation concentrations of L-glutamic acid are present in the presynaptic neuron; thus, increased substrate concentrations do not normally affect the rate of GABA synthesis. Therefore, the exogenous administration of L-glutamic acid may not significantly increase the neurotransmitter GABA, unless L-glutamic acid levels are abnormally low.
  • GABA-A receptor sensitive to the competitive blocking action of bicuculline and picrotoxin or picrotoxinin. These receptors are on postsynaptic structures and mediate classical inhibitory actions of GABA; and GABA-B receptors are located on presynaptic terminals and these receptors are insensitive to the blocker actions of bicuculline. GABA-B receptors can modify release of not only GABA in the CNS, but also NE from certain sites in the sympathetic nervous system.
  • GABA-modulin is similar to GTP regulator protein associated with receptors linked to adenylate cyclase. The activity of GABA-modulin is determined by phosphorylation.
  • GABA is typically associated with short inhibitory neurons in the hypothalamus, hippocampus, basal ganglia of the brain, substantia gelatinosa of the dorsal horn of the spinal cord and in the retina. Some long-axon pathways within the CNS have been identified as associated with GABAgeric activity.
  • GABA agonists include imidazole acetic acid, 3-aminopropane sulphonic acid, and THIP (4, 5, 6, 7, -tetrahydro-isoyazolo-[415-C]-pyridin-3-ol, and muscimol (3-hydroxy-5-amino-methylisoxazole) which is found in amanita muscaria.
  • GABA antagonists include bicuculline, picrotoxin, picrotoxinin and benzylpenicillin.
  • Inhibitors of GABA uptake include, for the neuronal uptake type, diaminobutyric acid and cis-2, 3aminocyclohexane, carboxylic acid; for the glial uptake type B-alanine; and for the miscellaneous uptake type, nipecotic acid, benzodiazepines, neuroleptics and tricyclic antidepressants.
  • GABA taken back into the presynaptic neuron after release and receptor interaction, is recycled as a potentially reusable transmitter.
  • GABA is enzymatically metabolized in both the nerve terminal and glial tissue and converted, in the presence of A-oxoglutamic acid, to succini semialdehyde by the mitochondrial enzyme GABA amino tranferase (GABA-T).
  • GABA-T GABA amino tranferase
  • the succinic acid which is formed enters the tricarboxylic acid (Krebs) cycle.
  • GABA-T requires pyridoxal phosphate as a co-factor.
  • Succinic semialdehyde is rapidly oxidized to succinic acid by the enzyme succinic semialdehyde dehydrogenase which also involves NAD and NADH as co-factors.
  • the inventors' formulation for RDS takes this fact into account by adding pyridoxal-5 -phosphate as a promoter ofthe oxidative-reductive pathway. In this regard.
  • GABA concentrations can be increased by the administration, to animals, including humans, of the following inhibitors of GABA-T: ethanoloamine-P-sulphate, gamma acetylenic GABA, gamma vinyl GABA, gabcuculline, hydazinopropionic acid, sodium di-N-propylacetate (sodium valproate) and aminooxyacetic acid (inhibitor of Vitamin B6), L-glutamine (Bloom, 1985).
  • Catecholamines are compounds which possess two adjacent hydroxyl (OH) groups on a benzene ring. In the body, such substances are synthesized form the aromatic amino acid L-tyrosine, which is hydroxylated to L-3, 4-dihydroxyphenylalanine (L-dopa) by the enzyme tyrosine hydroxylase. L-tyrosine is actively take up into noradrenergic nerve terminals. L-phenylalanine is a precursor of L-Tyrosine (Blum and Kozlowski, 1990; Schwartz et al, 1992).
  • Tyrosine hydroxylase is located in the cytoplasm of noradrenergic neurons and is the rate-limiting enzyme in the synthesis of NE.
  • Extensive research has revealed that reduced pteridine cofactor, molecular oxygen and ferrous ions are all required for activity.
  • L-dopa is decarboxylated to DA by L-aromatic amino acid decarboxylase, an enzyme which requires pyridoxal phosphate (Vitamin B6) as a cofactor.
  • the dopamine (DA) is actively taken up into granular storage vesicles in which the DA is hydroxylated to form norepinephrine (NE) by the enzyme dopamine-Bhydroxylase.
  • NE is further converted to epinephrine (E) by the enzyme phenylethanolamine-N-methyltransferase.
  • Tyrosine hydroxylase activity is influenced by the following: "end product" inhibition, caused by increased concentration of NE within nerve terminals which decreases the rate of conversion of L-tyrosine into L-dopa; increased sympathetic activity from the CNS which increases the synthesis of NE; the angiotensin II mediated increases the rate of NE synthesis; and agonists (e.g., clonidine) and blockers (e.g., phentolamine) of adreno-receptors which change the rate of NE release by mechanisms involving adrenergic receptors located on the presynaptic terminal.
  • end product inhibition caused by increased concentration of NE within nerve terminals which decreases the rate of conversion of L-tyrosine into L-dopa
  • increased sympathetic activity from the CNS which increases the synthesis of NE
  • the angiotensin II mediated increases the rate of NE synthesis
  • agonists e.g., clonidine
  • blockers e.
  • Inhibitors of the enzymes of NE synthesis include: methyl-p-tyrosine (inhibits tyrosine hydroxylase); carbidopa (inhibits aromatic amino acid decarboxylase in tissues outside the CNS); and diethyldithiocarbonate, FAI63 and disulfiram (inhibitors of dopamine-B-hydroxylase).
  • NE is stored within the nerve terminal in multiple storage complexes and more than one anatomical location.
  • One form of NE storage type is a granular complex found within vesicles in noradrenergic nerve terminals.
  • the granular complex consists of NE bound to ATP, several proteins collectively called chromogranins, includes dopamine-B-hydroxylase and Mg++, Zn++ and Cu++.
  • the uptake of DA and NE into storage vesicles is an active-transport process which requires ATP as an energy source and Mg++ to activate the ATPase enzyme which is Mg++ dependent.
  • This Mg++-dependent uptake process of NE and DA into storage vesicles is a separate and different process from the neuronal uptake process for NE across the nerve cell membrane, which is an Na.sup.+ /K.sup.+ -ATPase dependent.
  • the stability of the NE- ATP -protein- ion storage complex can be disrupted by some compounds which act as chelators of Mg++. This may be linked to the magnesium deficiency sometimes found in chronic cocaine abusers. In this regard, chronic administration of cocaine produces an increase in NE turnover.
  • NE from nerve terminals occurs by a process of exocytosis, which is calcium dependent, whereby a vesicular membrane fuses with the plasma membrane and the vesicular contents, consisting of NE, ATP, dopamine-Bhydroxylase and chromogranins, are released into the synaptic cleft.
  • exocytosis which is calcium dependent, whereby a vesicular membrane fuses with the plasma membrane and the vesicular contents, consisting of NE, ATP, dopamine-Bhydroxylase and chromogranins, are released into the synaptic cleft.
  • One mechanism known to control the availability of NE to postsynaptic receptors operates by means of presynaptic receptors located on the terminal from which NE is released.
  • the actions of NE in the synaptic cleft are terminated by removal from the synaptic cleft by an uptake system found on presynaptic nerve endings.
  • Uptake I is energy dependent, requiring ATP which is broken down by a sodium dependent ATPase. This is a high-affinity process, which means that it is efficient at the eliminating low concentrations of NE from the synaptic cleft.
  • the neuronal uptake system transports NE into the nerve terminal. Inside the nerve terminal most of the NE is taken up into storage vesicles. Inhibitors of this process include: cocaine, tricyclic anti-depressants, amphetamine and tyramine.
  • Uptake II involves the accumulation of NE by nonneuronal tissues.
  • High plasma levels of NE derived from stimulation of the adrenal medulla, or intravenous injection of a catecholamine will be removed by uptake into non-nervous tissues such as liver, muscle and connective tissue.
  • the NE or any other catecholamine diffuses back into the circulation or, more commonly is destroyed intracellularly by the enzymes monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT).
  • MAO monoamine oxidase
  • COMP catechol-O-methyltransferase
  • MAO is found in all tissues which contain mitochondria, and is bound to their outer membranes. MAO is present in liver, brain, nerves, muscles and all actively metabolizing tissues. It oxidatively deaminates NE to c, 4-dihydroxymandelic acid which can then by O-methylated (by COMT) to give rise to 3-methoxy-4-hydroxy-mandelic acid.
  • MAO in actuality describes a group of isoenzymes which possess different tissue distributions, substrate specificities, inhibitor characteristics and physical properties. For example, MAO A has a substrate preference for NE and 5HT, and is selectively inhibited by clorgyline.
  • MAO B has a substrate preference for olopamine and phenylethylamine, and is selectively inhibited by deprenyl (selegiline).
  • Other well known MAO inhibitors include iproniazid, nialamide, pargyline, tranclypromine and phenelzine.
  • COMT is found in large quantity in liver cells.
  • COMT acts on E and NE which has not been inactivated by neuronal re-uptake.
  • Pyrogallol an inhibitor works by blocking the COMT dependent transfer of a methyl group from S-adenosyl-L-methionine to the hydroxyl group at the 3' position of the catechol ring of NE, E and DA.
  • Dopamine is the precursor of NE and E, and plays a significant role in the CNS and at some ganglia in the autonomic nervous system. High intraneuronal amounts of DA inhibits tyrosine hydroxylase by end-product inhibition, thus decreasing the rate of DA synthesis.
  • the rate-limiting step in the synthesis of DA is the conversion of tyrosine to L-dopa by tyrosine hydroxylase.
  • tyrosine hydroxylase is completely saturated with L-tyrosine and thus increase in circulatory tyrosine levels do not increase the rate of DA synthesis.
  • this fact changes when there is a deficit in both the amount of DA and when tyrosine hydroxylase is compromised as under the influence of cocaine.
  • L-dopa is actively taken up into DA neurons in the CNS where it is converted to DA. Following L-dopa therapy there is a significantly increase in the amount of DA synthesized and stored. By comparison with the dopaminergic system, there is relatively little increase in the synthesis of NE following L-dopa, treatment.
  • Dopamine is stored in storage granules where the catecholamine is complexed with chromogranins, divalent metal ions and ATP.
  • DA is believed to be released into the synaptic cleft by exocytosis. As with NE, this is a calcium dependent process and occurs in response to action potentials reaching nerve terminals or to drugs.
  • the following substances can increase DA release; cocaine, (+)-amphetamine, methylamphetamine, tyramine, amantadine, m-phenmetrazine, phentermine and nomifensine.
  • these compounds can also, to different degrees, inhibit neuronal re-uptake of DA.
  • DA neuronal re-uptake system which is a high affinity, energy-dependent active-transport process.
  • MAO and COMT are responsible for the transformation of DA to 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA, 3-methoxy-4-hydroxy-phenylacetic acid), respectively.
  • DOPAC 4-dihydroxyphenylacetic acid
  • HVA homovanillic acid
  • Cocaine by virtue of blocking re-uptake of DA into presynaptic nerve terminals, prolongs the effect of release DA in the synaptic cleft.
  • Elevation of brain tyrosine levels results in an increase in L-DOPA synthesis in the brain.
  • L-DOPA in turn is metabolized to dopamine.
  • the synthesis and release of dopamine is elevated following tyrosine administration.
  • dietary tyrosine increases turnover and release of dopamine and norepinephrine.
  • Stress, cold or certain drugs induce an increase in nerve firing to lower the levels of catecholamines in the nerve terminals.

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  • Wood Science & Technology (AREA)
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  • Inorganic Chemistry (AREA)
  • Zoology (AREA)
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  • Obesity (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
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Abstract

La présente invention concerne une amélioration du traitement de l'attention obtenu par administration d'un inhibiteur d'endorphinase ou d'un inhibiteur d'enképhalinase et éventuellement, d'un précurseur de dopamine ou d'un précurseur de sérotonine, d'un précurseur de GABA ou d'un agent de libération d'endorphine ou d'enképhalinase, ou de certains composés d'origine végétale comprenant de l'extrait de rose Rhodiola (Pharmaline) et/ou Huperzine. Ces composants favorisent la restauration d'une fonction normale de neurotransmetteur et les composants associés améliorent la libération de dopamine au niveau du noyau accumbens et ne présentent pas d'accoutumance. On préfère, en particulier, l'utilisation de précurseurs de dopamine, L-phénylalanine, ou L-Tyrosine, de l'inhibiteur d'enképhalinase, D-phénylalanine, et/ou le précurseur de sérotonine, 5-hydroxytryptophane, et d'un inhibiteur naturel d'acétylcholénestérase et de sels de chrome (c'est-à-dire, picolinate, nicotinate, etc. ), cette utilisation n'étant pas limitée et contribuant au soulagement des symptômes associés à une déficience en phénylalanine du cerveau.
PCT/US1998/008684 1997-04-29 1998-04-29 Diagnostic d'un syndrome d'insatisfaction a l'aide de polygene allelique et traitement associe Ceased WO1998048785A2 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
AU72677/98A AU7267798A (en) 1997-04-29 1998-04-29 Allelic polygene diagnosis of reward deficiency syndrome and treatment
EP98920019A EP0979092A2 (fr) 1997-04-29 1998-04-29 Diagnostic d'un syndrome d'insatisfaction a l'aide de polygene allelique et traitement associe
IL13263498A IL132634A0 (en) 1997-04-29 1998-04-29 Allelic polygene diagnosis of reward deficiency syndrome and treatment
JP54736498A JP2002511850A (ja) 1997-04-29 1998-04-29 報酬欠損症候群の対立遺伝子多遺伝子診断および処置
CA002288990A CA2288990A1 (fr) 1997-04-29 1998-04-29 Diagnostic d'un syndrome d'insatisfaction a l'aide de polygene allelique et traitement associe
NO995257A NO995257L (no) 1997-04-29 1999-10-28 Fremgangsmåte og materialer for diagnose og behandling av Reward Deficiency Syndrome
IS5233A IS5233A (is) 1997-04-29 1999-10-28 Greining á fjölgenesamsætum tengdum umbunarskortsheilkenni (reward deficiency syndrome) og efnablöndum til meðferðar við því

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US4439497P 1997-04-29 1997-04-29
US60/044,394 1997-04-29

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CA (1) CA2288990A1 (fr)
IL (1) IL132634A0 (fr)
IS (1) IS5233A (fr)
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US6132724A (en) * 1998-04-29 2000-10-17 City Of Hope National Medical Center Allelic polygene diagnosis of reward deficiency syndrome and treatment
WO2001038567A3 (fr) * 1999-11-24 2002-03-21 Isis Innovation Indicateurs genetiques de consommation de tabac
WO2002085341A3 (fr) * 2001-04-23 2003-05-08 Sigma Tau Healthscience Spa Composition pour la prevention ou le traitement de troubles d'apprentissage chez les enfants souffrant de trouble d'hyperactivite avec deficit de l'attention
US6955873B1 (en) * 2000-08-04 2005-10-18 Kenneth Blum Diagnosis and treatment system for reward deficiency syndrome (RDS) and related behaviors
WO2010005303A3 (fr) * 2008-07-07 2010-04-29 Publiekrechtelijke Rechtspersoon Academisch Ziekenhuis Leiden H.O.D.N. Leids Universitair Medisch Centrum Nouveaux indicateurs de longévité humaine et du taux de vieillissement biologique
JP2011172942A (ja) * 2000-02-09 2011-09-08 Cns Response Inc 定量的ｅｅｇを使用して脳の生理学的不均衡を分類および処置する方法
US8142800B1 (en) * 2001-12-18 2012-03-27 Tamea Rae Sisco Oral high potency clinical anti-craving treatment and method of use
US8142799B2 (en) * 2001-12-18 2012-03-27 Tamea Rae Sisco High potency clinical anti-craving treatment and method of use
EP2485747A4 (fr) * 2008-06-21 2013-10-23 Kenneth Blum Personnalisation dirigée par l'adn des composés analgesiques comme modalité thérapeutique
US8572472B2 (en) 2006-09-28 2013-10-29 Mitsubishi Electric Corporation Fault detection apparatus, fault detection method, and fault detection program
EP2585824A4 (fr) * 2010-04-22 2014-01-01 Kenber Llc Analyse du risque génétique dans le syndrome du déficit de récompense
CN103990106A (zh) * 2014-06-11 2014-08-20 河北医科大学 八肽胆囊收缩素在制药中的应用
CN104846015A (zh) * 2015-05-27 2015-08-19 深圳先进技术研究院 特异性兴奋伏隔核中的gaba能神经元的组合物及其在改善精神分裂症异样行为中的应用
EP3156053A1 (fr) * 2008-06-20 2017-04-19 Afecta Pharmaceuticals, Inc. Utilisation d'isoindoles pour le traitement de troubles de comportement neurologique
US9727691B2 (en) 2011-09-30 2017-08-08 Tokyo Metropolitan Institute Of Medical Science Method for evaluating drug sensitivity and disease vulnerability by analyzing cyclic AMP responsive element binding protein gene
CN113332299A (zh) * 2021-05-27 2021-09-03 广东湛江海洋医药研究院 红景天苷在缓解糖皮质激素性骨质疏松症中的应用
US11464756B1 (en) 2017-05-19 2022-10-11 Jerry Darm Mecuna pruriens, L-DOPA and 5-HTP based dietary supplements, pharmaceutical formulations and uses thereof

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WO2006086242A2 (fr) * 2005-02-07 2006-08-17 Genenews, Inc. Biomarqueurs de l'osteoarthrite benigne et utilisations
EP2552536B1 (fr) 2010-04-01 2016-06-08 Ecole Polytechnique Fédérale de Lausanne (EPFL) Dispositif d'interaction avec un tissu neurologique
JP2015067581A (ja) * 2013-09-30 2015-04-13 国立大学法人京都大学 多重共鳴用のプローブ
US11311718B2 (en) 2014-05-16 2022-04-26 Aleva Neurotherapeutics Sa Device for interacting with neurological tissue and methods of making and using the same
US9925376B2 (en) * 2014-08-27 2018-03-27 Aleva Neurotherapeutics Treatment of autoimmune diseases with deep brain stimulation
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US10702692B2 (en) 2018-03-02 2020-07-07 Aleva Neurotherapeutics Neurostimulation device
DE102018114364A1 (de) 2018-06-15 2019-12-19 Susanne Koch Verfahren und Vorrichtungen zur Bereitstellung eines Parameters, der auf eine erhöhte Wahrscheinlichkeit des Auftretens eines postoperativen Delirs hinweist
CN109316196B (zh) * 2018-12-12 2022-07-19 上海市精神卫生中心（上海市心理咨询培训中心） 基于虚拟现实联合脑电监测的苯丙胺类兴奋剂渴求评估方法
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US4761429A (en) * 1985-07-22 1988-08-02 Kenneth Blum Enkephalinase and endorphinase inhibitors as anti-craving compositions
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US5019594A (en) * 1989-11-28 1991-05-28 Interneuron Pharmaceuticals, Inc. Method for decreasing appetite
US5164384A (en) * 1991-06-19 1992-11-17 Metagenics, Inc. Anabolic mineral formula
US5543405A (en) * 1993-10-22 1996-08-06 Keown; Wendy J. Composition and method for weight reduction and long term management of obesity

Cited By (19)

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US6132724A (en) * 1998-04-29 2000-10-17 City Of Hope National Medical Center Allelic polygene diagnosis of reward deficiency syndrome and treatment
WO2001038567A3 (fr) * 1999-11-24 2002-03-21 Isis Innovation Indicateurs genetiques de consommation de tabac
JP2003514571A (ja) * 1999-11-24 2003-04-22 アイシス・イノベーション・リミテッド タバコ消費の遺伝子インジケーター
US7745112B1 (en) 1999-11-24 2010-06-29 g-Nostics Limited Genetic indicators of tobacco consumption
JP2011172942A (ja) * 2000-02-09 2011-09-08 Cns Response Inc 定量的ｅｅｇを使用して脳の生理学的不均衡を分類および処置する方法
US6955873B1 (en) * 2000-08-04 2005-10-18 Kenneth Blum Diagnosis and treatment system for reward deficiency syndrome (RDS) and related behaviors
WO2002085341A3 (fr) * 2001-04-23 2003-05-08 Sigma Tau Healthscience Spa Composition pour la prevention ou le traitement de troubles d'apprentissage chez les enfants souffrant de trouble d'hyperactivite avec deficit de l'attention
US8142800B1 (en) * 2001-12-18 2012-03-27 Tamea Rae Sisco Oral high potency clinical anti-craving treatment and method of use
US8142799B2 (en) * 2001-12-18 2012-03-27 Tamea Rae Sisco High potency clinical anti-craving treatment and method of use
US8572472B2 (en) 2006-09-28 2013-10-29 Mitsubishi Electric Corporation Fault detection apparatus, fault detection method, and fault detection program
EP3156053A1 (fr) * 2008-06-20 2017-04-19 Afecta Pharmaceuticals, Inc. Utilisation d'isoindoles pour le traitement de troubles de comportement neurologique
EP2485747A4 (fr) * 2008-06-21 2013-10-23 Kenneth Blum Personnalisation dirigée par l'adn des composés analgesiques comme modalité thérapeutique
WO2010005303A3 (fr) * 2008-07-07 2010-04-29 Publiekrechtelijke Rechtspersoon Academisch Ziekenhuis Leiden H.O.D.N. Leids Universitair Medisch Centrum Nouveaux indicateurs de longévité humaine et du taux de vieillissement biologique
EP2585824A4 (fr) * 2010-04-22 2014-01-01 Kenber Llc Analyse du risque génétique dans le syndrome du déficit de récompense
US9727691B2 (en) 2011-09-30 2017-08-08 Tokyo Metropolitan Institute Of Medical Science Method for evaluating drug sensitivity and disease vulnerability by analyzing cyclic AMP responsive element binding protein gene
CN103990106A (zh) * 2014-06-11 2014-08-20 河北医科大学 八肽胆囊收缩素在制药中的应用
CN104846015A (zh) * 2015-05-27 2015-08-19 深圳先进技术研究院 特异性兴奋伏隔核中的gaba能神经元的组合物及其在改善精神分裂症异样行为中的应用
US11464756B1 (en) 2017-05-19 2022-10-11 Jerry Darm Mecuna pruriens, L-DOPA and 5-HTP based dietary supplements, pharmaceutical formulations and uses thereof
CN113332299A (zh) * 2021-05-27 2021-09-03 广东湛江海洋医药研究院 红景天苷在缓解糖皮质激素性骨质疏松症中的应用

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CA2288990A1 (fr) 1998-11-05
AU7267798A (en) 1998-11-24
CN1261801A (zh) 2000-08-02
KR20010020422A (ko) 2001-03-15
EP0979092A2 (fr) 2000-02-16
IL132634A0 (en) 2001-03-19
WO1998048785A3 (fr) 1999-04-01
NO995257L (no) 1999-12-27
WO1998048785A9 (fr) 1999-05-20
NO995257D0 (no) 1999-10-28
JP2002511850A (ja) 2002-04-16
IS5233A (is) 1999-10-28

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