WO2001001980A9

WO2001001980A9 - Methods for the amelioration of neuropsychiatric disorders by inhibiting the inactivating transport of endogenous cannabinoid substances

Info

Publication number: WO2001001980A9
Application number: PCT/US2000/018613
Authority: WO
Inventors: Daniele Pionnelli
Original assignee: Univ California; Daniele Pionnelli
Priority date: 1999-07-06
Filing date: 2000-07-06
Publication date: 2002-08-29
Also published as: AU6605500A; WO2001001980A1

Abstract

The invention is directed to a method for ameliorating a neuropsychiatric disorder in a patient by inhibiting the inactivating transport of an endogenous cannabinoid substance. The method comprises administration of a pharmaceutical composition able to inhibit the transport of an endogenous cannabinoid substance into cells. The administration is in an amount sufficient to inhibit the inactivating transport of an endogenous cannabinoid substance and to ameliorate the neuropsychiatric disorder in the patient.

Description

METHODS FOR THE AMELIORATION OF

NEUROPSYCHIATRIC DISORDERS BY INHIBITING

THE INACTIVATING TRANSPORT OF ENDOGENOUS

CANNABINOID SUBSTANCES

RELATED APPLICATIONS

This application incorporates by reference and claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 60/142,619, filed July 06, 1999. The aforementioned application is explicitly incorporated herein by reference in its entirety and for all purposes.

STATEMENTAS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under a grant from National Institutes of Health. The Government may have certain rights in this invention.

TECHNICAL FIELD This invention relates to the fields of neuroscience and psychiatry. In particular, the invention relates to methods for ameliorating neuropsychiatric disorders by inhibiting the inactivating transport of an endogenous cannabinoid substance.

BACKGROUND Functional hyperactivity of dopamine neurotransmission in the central nervous system (CNS) is thought to contribute to a diversity of neuropsychiatric conditions, which include schizophrenia, attention-deficit hyperactivity disorder, borderline personality disorder, autism and Tourette's syndrome. Thus, current treatments of these conditions are based on the use of dopamine D₂-type receptor antagonists. These drugs are, however, only partially effective and cause a wide variety of serious side effects, such as sedation and dizziness. Thus, there exists a need for alternative compounds that can counteract the neuropsychiatric conditions caused or aggravated by dopamine hyperactivity. SUMMARY

The invention provides a method for ameliorating a neuropsychiatric disorder in a patient in need thereof by inhibiting the inactivating transport of an endogenous cannabinoid substance, wherein the method comprises administration to the patient of a pharmaceutical composition able to inhibit the inactivating transport of an endogenous cannabinoid into cells, wherein the administration of the pharmaceutical composition is in an amount sufficient to inhibit the inactivating transport of an endogenous cannabinoid substance and to ameliorate the neuropsychiatric disorder in the patient. In alternative embodiments, the endogenous cannabinoid substance comprises anandamide and 2-arachidonylglycerol (2-AG). In the methods of the invention, the pharmaceutical composition can comprise a compound consisting essentially of (i) a hydrophobic carbon chain moiety comprising at least one nonconjugated cis double bond in the middle of the chain, linked to (ii) a polar carboxamido or carboxyester moiety, linked to (iii) a polar nonionizable head group. The hydrophobic carbon chain moiety can have one to six nonconjugated cis double bonds. The hydrophobic carbon chain moiety can have a length of C-18 to C-22. The compound can be compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 13, compound 20 or compound 21 or an equivalent thereof or a mixture thereof, the compounds as described in Figures 1 and 2.

In alternative embodiments, the pharmaceutical composition comprises N-(4-hydroxyphenyl) arachidonamide (AM404), N-(3-hydroxyphenyl) arachidonamide or an equivalent thereof or a mixture thereof. The polar head group can further comprise an alkyl group in the form of an S isomer. The pharmaceutical composition can comprise an S-l'-methyl anandamide (compound 21, Figure 2).

The pharmaceutical composition can comprise a compound consisting essentially of (i) a hydrophobic carbon chain moiety comprising at least one nonconjugated cis double bond in the middle of the chain, linked to (ii) a polar carboxamido or carboxyester moiety, linked to (iii) a head group as set forth in compound 11, compound 12, compound 18, compound 19, compound 20, compound 21, compound 22, compound 23, compound 28, compound 29, compound 30, compound 31, compound 32, compound 33 or compound 34 or an equivalent thereof or a mixture thereof (Figure 2). The hydrophobic carbon chain moiety can have one to six nonconjugated cis double bonds. The hydrophobic carbon chain moiety can have a length of C-l 8 to C-22. The head group can be a polar nonionizable head group with a hydrogen-donating hydroxyl group. Alternatively, the head group can have a polar nonionizable head group with a hydrogen-accepting group. The hydrogen-accepting group can be an ether containing group or a phenolic group.

In one embodiment, the pharmaceutical composition can comprise oleylethanolamide or oleamide or an equivalent thereof or a mixture thereof.

In one embodiment, inhibiting the inactivating transport can comprise inhibiting the inactivating transport of an endogenous cannabinoid substance. Inhibiting the inactivating transport of an endogenous cannabinoid substance can cause accumulation of the endogenous cannabinoid substance at its site of action.

In another embodiment, inhibiting the inactivating transport of an endogenous cannabinoid substance can counteract the effects of dopamine hyperactivity. The neuropsychiatric disorder ameliorated by the methods of the invention can be at least in part caused or mediated by dopamine hyperactivity. The dopamine hyperactivity can be in a central nervous system (CNS) region.

In alternative embodiments, the neuropsychiatric disorder is a schizophrenia, a schizoaffective disorder, a schizophreniform disorder, a borderline personality disorder, an attention-deficit hyperactivity disorder, an autism spectrum disorder, Tourette's syndrome, and a psychoactive substance-induced organic mental disorder or a psychoactive substance use disorder.

In one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable excipient comprising an aqueous solution or a lipid based solution. The pharmaceutical composition can be administered by an oral, a parenteral, a sublingual, a transmucosal or a transdermal route, for example.

The invention also provides a pharmaceutical composition comprising a compound able to inhibit the inactivating transport of an endogenous cannabinoid substance and a pharmaceutically acceptable excipient. The pharmaceutical composition can consist essentially of (i) a hydrophobic carbon chain moiety comprising at least one nonconjugated cis double bond in the middle of the chain, linked to (ii) a polar carboxamido or carboxyester moiety, linked to (iii) a polar nonionizable head group. The hydrophobic carbon chain moiety can have one to six nonconjugated cis double bonds. The hydrophobic carbon chain moiety can have a length of C-l 8 to C-22. The compound can be compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 13, compound 20 or compound 21 or an equivalent thereof or a mixture thereof (see Figures 1 and 2). The compound can comprise N-(4-hydroxyphenyl)arachidonamide (AM404), N-(3- hydroxyphenyl)arachidonamide or an equivalent thereof or a mixture thereof. The pharmaceutical composition can have a hydroxyl ethyl head group further comprising an alkyl group in the form of an S isomer. The pharmaceutical composition can comprise an S-l'-methyl anandamide (compound 21). In alternative embodiments, the compound consists essentially of (i) a hydrophobic carbon chain moiety comprising at least one nonconjugated cis double bond in the middle of the chain, linked to (ii) a polar carboxamido or carboxyester moiety, linked to (iii) a head group as set forth in compound 11, compound 12, compound 18, compound 19, compound 20, compound 21, compound 22, compound 23, compound 28, compound 29, compound 30, compound 31, compound 32, compound 33 or compound 34 or an equivalent thereof or a mixture thereof. The pharmaceutical composition can have a hydrophobic carbon chain moiety having one to six nonconjugated cis double bonds. The hydrophobic carbon chain moiety can have a length of C-l 8 to C-22. The head group can be a polar nonionizable head group with a hydrogen-donating hydroxyl group. Alternatively, the head group can be a polar nonionizable head group with a hydrogen-accepting group. The hydrogen-accepting group can be an ether containing group or a phenolic group. The compound can comprise an oleyl-ethanolamide or an oleamide or an equivalent thereof or a mixture thereof. In one embodiment, the concentration of the compound of the pharmaceutical composition in the pharmaceutically acceptable excipient is between about 0.1 mg per kg and about 10 mg per kg of body weight. The pharmaceutically acceptable excipient can be an aqueous solution or a lipid- (e.g., oil-) based solution. The pharmaceutical composition can be formulated for administration by an oral, a parenteral, a sublingual, a transmucosal or a transdermal route. The invention also provides a kit comprising a pharmaceutical composition and printed material, wherein the pharmaceutical composition comprises a compound and a pharmaceutically acceptable excipient, wherein the compound is able to inhibit the inactivating transport of an endogenous cannabinoid substance, and wherein the printed matter comprises instructions for use of the pharmaceutical composition to ameliorate a neuropsychiatric disorder in a patient in need thereof.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences and ATCC deposits cited herein are hereby expressly incorporated by reference for all purposes.

DESCRIPTION OF DRAWINGS Figure 1 illustrates fatty acid ethanolamides used as inhibitors of radio labeled anandamide uptake by tissue culture cells and shows the results of these experiments (right-hand column) where IC₅₀ values in M are expressed and the mean +/- SEM of three independent experiments conducted in triplicate.

Figure 2 illustrates anandamide analogs containing carboxamide and polar head group modifications used as inhibitors of radiolabeled anandamide uptake by tissue culture cells and shows the results of these experiments (right-hand column) where IC50 values in M are expressed and the mean +/- SEM of three independent experiments conducted in triplicate.

Figure 3 illustrates schematic low-energy conformers of various fatty acid ethanolamides with hydrophobic carbon chains differing in their degree of unsaturation. The numbers indicate calculated interatomic distances in A. (A) anandamide; (B) cw-eicosatrienoylethanolamide (2 of Figure 1, 20:3Δ⁸'^π'¹⁴); (C) cis- eicosadienoylethanolamide (3 of Figure 1, 20:2Δⁿ'¹⁴); (D) cis- eicosaenoylethanolamide (4 of Figure 1, 20:lΔ^π); (E) oleylethanolamide (6 of Figure 1, 18:1 Δ⁹); (F) trarcs-octadecenoylethanolamide (7 of Figure 1, 18:1 Δ⁹).

Like reference symbols in the various drawings indicate like elements. DETAILED DESCRIPTION

The invention provides novel methods for ameliorating neuropsychiatric disorders, particularly including those at least in part caused or mediated by dopamine hyperactivity, including, e.g., schizophrenia, schizoaffective disorder, schizophreniform disorder, borderline personality disorder, attention-deficit hyperactivity disorder, autism spectrum disorder, Tourette's syndrome or a psychoactive substance-induced organic mental disorder or a psychoactive substance use disorder. In the methods of the invention, a pharmaceutical composition is administered that inhibits the inactivating transport of an endogenous cannabinoid substance, including, e.g., anandamide or 2-arachidonylglycerol (2-AG). The inactivating transport of the endogenous cannabinoid is inhibited by, e.g., inhibiting the inactivating transport of the endogenous cannabinoid from an extracellular space, e.g., a synaptic space. The administration of the pharmaceutical composition is in an amount sufficient to inhibit the inactivating transport of the endogenous cannabinoid substance, thereby ameliorating the neuropsychiatric disorder in the patient.

The invention has established that a prominent functional role of the endogenous cannabinoid system is to counteract and modulate dopamine hyperactivity. While the invention is not dependent or based on any particular mechanism of action, pharmacological inhibitors of endogenous cannabinoid inactivation can antagonize (i.e., oppose the action of, or ameliorate the results of) dopamine hyperactivity. The pharmacological inhibitors can be inhibitors of inactivating transport of endogenous cannabinoid substances and cause the accumulation of endogenous cannabinoids at their synaptic sites of action. As described herein, the methods of the invention have been demonstrated to ameliorate neuropsychiatric disorders by using art-accepted animal models (see Example 1). Specifically, the pharmacological properties of the anandamide transport inhibitor N-(4-hydroxyphenyl)-arachidonamide (AM404) was characterized in rats. The effects of this drug was investigated by using various behavioral responses associated with activation of dopamine D₂-family receptors. Rat brain slices accumulated [ H] anandamide via a high-affinity transport mechanism that was blocked by AM404. When administered alone in vivo, AM404 caused a mild and slow-developing hypokinesia that was significant 60 minutes (min) after intracerebro entricular injection of the drug. This hypokinesia was reversed by the CB1 cannabinoid receptor antagonist SR141716A. AM404 produced no significant catalepsy or analgesia, two typical effects of direct-acting cannabinoid agonists. However, AM404 prevented the stereotypic yawning produced by systemic administration of a low dose of apomorphine, an effect that was dose-dependent and blocked by SR141716A. Furthermore, AM404 reduced the stimulation of motor behaviors elicited by the selective D -family receptor agonist quinpirole. Finally, AM404 reduced hyperactivity in juvenile spontaneously hypertensive rats, a putative model of attention-deficit hyperactivity disorder. The results support a primary role of the endocannabinoid system in the regulation of psychomotor activity. They also demonstrate that anandamide transport is a target for neuropsychiatric medicines.

In summary, the methods of the invention have been demonstrated to ameliorate neuropsychiatric disorders by determining the behavioral effects of one of the exemplary compounds that can be used in the methods of the invention, AM404. AM404 is a selective inhibitor of endogenous cannabinoid transport. Two art- recognized animal models were used that are predictive of antipsychotic activity: (1) the motor hyperactivity induced by dopamine agonist (quinpirole) in rats, and (2) the yawning response induced by the dopamine agonist apomorphine in rats. In both models, AM404 produced a highly effective inhibition of dopamine agonist-induced hyperactivity, when injected at intraperitoneal doses of 10 to 20 mg/kg, or intracerebro ventricular doses of 2 to 10 μg/rat. Importantly AM404 had little effect when administered alone, indicating that its actions are selectively expressed during dopamine hyperactivity, a feature that is highly desirable therapeutically.

The biological actions of the endogenous cannabinoid anandamide are terminated by carrier-mediated transport into neurons and astrocytes, followed by enzymatic hydrolysis. Anandamide transport is inhibited by the compound AM404. AM404 does not bind productively to CB1 cannabinoid receptors, but potentiates several responses elicited by administration of exogenous anandamide. The findings of the invention demonstrate that AM404 protects endogenous anandamide from inactivation. The effects of AM404 administration on the plasma levels of anandamide and other fatty acid ethanolamides (palmitylethanolamide, PEA, and oleylethanolamide,OEA) using HPLC/MS were tested to demonstrate the in vivo efficacy of inhibitors of endogenous cannabinoid inactivation (see Example 2, below). Systemic administration of AM404 (10 mg-kg^"1) caused a gradual increase of anandamide in plasma, which was significantly different from untreated controls at 60 and 120 min after drug injection. By contrast, AM404 had no effects on plasma PEA levels, and increased OEA levels only 120 min after injection. AM404 caused a time- dppendent decrease of motor activity, which was reversed by the CB1 antagonist SR141716A.

Thus, the invention provides methods for the treatment of neuropsychiatric conditions characterized by excessive dopamine activity. These methods are based on the use of pharmacological agents that inhibit the inactivating transport of anandamide and 2-arachidonylglycerol (2-AG), two endogenous cannabinoid substances.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meamng commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

The term "AM404" refers to N-(4-hydroxyphenyl) arachidonamide and stractoal/functional equivalents thereof, including, e.g., N-(3-hydroxyphenyl) arachidonamide. See, e.g., Piomelli (1999) Proc. Natl. Acad. Sci. 96:5802-5807; Beltramo (1997) Science 277:1094-1097, as described in detail, below.

The phrase "administration of a pharmaceutical composition" incorporates the phrases common usage and refers to any appropriate means to give a pharmaceutical to a patient, taking into consideration the properties of the pharmaceutical composition and the preferred site of administration; e.g. , in one embodiment, the pharmaceutical composition of the invention is injected into the epidural or the subarachnoid space.

The term "ameliorating" as used herein refers to any indicia of success in the treatment or amelioration of an injury, pathology, condition, or symptom (e.g., neuropsychiatric disorder), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the symptom, injury, pathology or condition more tolerable to the patient; decreasing the frequency or duration of the symptom or condition; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient's physical or mental well-being; or, in some situations, preventing the onset of the symptom or condition, e.g., a neuropsychiatric disorder. The treatment or amelioration of symptoms can be based on any objective or subjective parameter; including, e.g. , the results of a physical examination and/or a psychiatric evaluation, or, simply an improvement in the patient's sense of well-being. For example, the methods of the invention ameliorate neuropsychiatric disorders involving dopamine hyperactivity, including, e.g., schizophrenia, attention-deficit hyperactivity disorder, borderline personality disorder, autism and Tourette's syndrome.

The term "anandamide" as used herein refers to arachidonylethanolamides or equivalents (see, e.g., U.S. Patent No. 5,631,297) that are the same or equivalent to endogenous lipids that activate brain cannabinoid receptors and mimic the pharmacological effects of 9-tetrahydrocannabinol, the active principle of hashish and marijuana, as described in further detail, below.

Anandamide analogs (equivalents) are described, e.g., in U.S. Patent No. 5,977,180;

WO 99/60987; WO 99/64389. See also, e.g., U.S. Patent Nos. 6,028,084; 6,013,648;

5,990,170; 5,925,672; 5,747,524; 5,596,106; EP 0 570920, WO 94-12466.

The terms "2-arachidonylglycerol" or "2-AG" as used herein refer to those compounds and their equivalents which are endogenous agonists of brain cannabinoid receptors. See, e.g., Sugiura (2000) Ann. NY Acad. Sci. 905:344-346;

Sugiura (1996) Biochem. Biophys. Res. Commun. 229:58-64; Di Marzo (1996J

Biochem. Biophys. Res. Commun. 227:281-288; Lee (1995; J. Pharmacol. Exp. Ther.

275:529-536. The term "endogenous cannabinoid substance" as used herein refers to an endogenous agonist, or an equivalent thereof, of a cannabinoid receptor.

Cannabinoid receptors are described, e.g., in U.S. Patent No. 6,013,648. Endogenous agonists include, e.g., 2-arachidonylglycerol or anandamide . See also U.S. Patent

Nos. 6,028,084; 6,017,919; 596,106; 5,990,170; and, Seltzman (1999) Curr. Med. Chem. 6:685-704. The term "neuropsychiatric disorder" as used herein refers to any neuropsychiatric disorder that can be ameliorated by administration of a pharmaceutical composition able to inhibit the inactivating transport (e.g., the intracellular transport) of an endogenous cannabinoid substance. Specific neuropsychiatric disorders as used herein are as defined by the Diagnostic and

Statistical Manual of Mental Disorders, Fourth Edition (DSM-IN) (1994) Task Force on DSM-IN, American Psychiatric Association; see also, Kaplan, Ed. (1995), Comprehensive Textbook of Psychiatry, vol. 1, sixth edition, Williams & Williams, Baltimore MD. While the invention is not limited by any particular mechanism of action, in one embodiment, inhibiting the inactivating transport of an endogenous cannabinoid substance counteracts the effects of dopamine hyperactivity, as described in further detail, below.

The phrase "inhibiting the inactivating transport of an endogenous cannabinoid substance by administration of a pharmaceutical composition" means any measurable amount of increase in the amount of extracellular free endogenous cannabinoid substance, e.g., an increase in the amount of free endogenous cannabinoid substance in a synaptic space. While the invention is not limited by any specific mechanism, the inactivating transport can be accomplished by the pharmaceutical composition by inhibition of inactivating uptake of the endogenous cannabinoid substance by the cell membrane (e.g., synaptic membrane).

The term "pharmaceutically acceptable excipient" incorporates the common usage and includes any suitable pharmaceutical excipient, including, e.g., water, saline, phosphate buffered saline, Hank's solution, Ringer's solution, dextrose/saline, glucose, lactose, or sucrose solutions, magnesium stearate, sodium stearate, glycerol monostearate, glycerol, propylene glycol, ethanol, and the like.

The term "subarachnoid space" or cerebral spinal fluid (CSF) space incorporates the common usage and refers to the anatomic space between the pia mater and the arachnoid membrane containing CSF. General Methods

The methods of the invention use compounds capable of ameliorating a neuropsychiatric disorder by inhibiting the inactivating transport of an endogenous cannabinoid substance. A variety of exemplary compounds useful in these methods are described herein. However, any compound which can inhibit the inactivating transport of an endogenous cannabinoid substance, including an equivalent of the exemplary compounds described herein, is envisioned to be useful in the methods of the invention. Exemplary routine methods for identifying these compounds are described herein. The skilled artisan will recognize that compounds useful in the methods of the invention (e.g., arachidonylethanolamide or 2-arachidonylglycerol) can be synthesized using a variety of procedures and methodologies, which are well described in the scientific and patent literature., e.g., Organic Syntheses Collective Volumes, Gilman et al. (Eds) John Wiley & Sons, Inc., NY; Nenuti (1989) Pharm Res. 6:867-873. The invention can be practiced in conjunction with any method or protocol known in the art, which are well described in the scientific and patent literature. Therefore, only a few general techniques will be described prior to discussing specific methodologies and examples relative to the methods of the invention. Structural Guidelines and Screening Tests to Design Inhibitors of Endogenous Cannabinoid Inactivating Transport Useful in the Methods of the Invention h the methods of the invention, pharmaceutical compositions comprising compounds capable of ameliorating a neuropsychiatric disorder are administered. These compounds act by inhibiting the inactivating transport of an endogenous cannabinoid substance. In one embodiment of the invention, a compound capable of ameliorating a neuropsychiatric disorder inhibits the inactivating transport of anandamide, an endogenous cannabinoid substance. An exemplary inhibitory compound is Ν-(4-hydroxyphenyl)-arachidonamide (AM404), which is a structural analog of anandamide. AM404 is characterized by a highly hydrophobic carbon chain and a polar carboxamido group carrying a hydroxyphenyl moiety.

The biological actions of anandamide (arachidonyl-ethanolamide), an endogenous cannabinoid lipid, can be terminated by a two-step inactivation process consisting of carrier-mediated uptake and intracellular hydrolysis. Anandamide uptake in neurons and astrocytes is mediated by a high-affinity, Na⁺-independent transporter. This uptake can be selectively inhibited, e.g., by AM404 or equivalents. Equivalent compounds that can be used in the methods of the invention can be determined by analysis of structural determinants governing recognition and translocation of substrates by the anandamide transporter. One useful model measures translocation of compounds by anandamide transporter constitutively expressed in a human astrocytoma cell line, as described herein (see also, e.g., Piomelli (1999) supra). Competition experiments revealed that substrate recognition by the transporter is favored by a polar non-ionizable head group of defined stereochemical configuration containing a hydroxyl moiety at its distal end. The secondary carboxamide group interacts favorably with the transporter, but may be replaced with either a tertiary amide or an ester, suggesting that it serves as hydrogen acceptor. As a result, 2-arachidonylglycerol, an endogenous cannabinoid ester, can also serve as a transport substrate. Substrate recognition requires also the presence of at least one cis double bond situated at the middle of the fatty acid carbon chain, indicating a preference for ligands whose hydrophobic tail can adopt a bent U-shaped conformation. On the other hand, uptake experiments with radioactively labeled substrates show that four cis non-conjugated double bonds are a minimum requirement for translocation across the cell membrane, suggesting that substrates are transported in a folded hairpin conformation (however, a compound can successfully inhibit the inactivating transport of an endogenous cannabinoid substance without comprising four cis non-conjugated double bonds or being translocated across a cell membrane). These results outline the general structural requisites for anandamide transport and provides the requisite screening parameters in the selection of compounds useful in the methods of the invention.

All amides can be synthesized by the reaction of the fatty acid or fatty acid chloride with the appropriate amine or aminoalcohol, as described by, e.g., Abadjj (1994) J. Med. Chem. 37:1889-1893. 1- and 2-arachidonylglycerols can be prepared by a modification of the procedure established by Serdarevich (1966) J.

Lipid Res. 7:277-284, for the synthesis of fatty acid monoglycerides. Briefly, 1,3-0- benzylidine--f«-glycerol, prepared by the reaction of glycerol with benzaldehyde in the presence of p-toluenesulfonic acid, was allowed to react with arachidonyl chloride in the presence of pyridine. Subsequent treatment with boric acid in triethylborate to remove the benzylidine moiety gave 2-arachidonylglycerol. For the preparation of l(3)-arachidonylglycerol, commercially available l,2-O-isopropylidene-5.-glycerol 5 was esterified with arachidonic acid in a similar manner, followed by removal of the isopropylidene group by treatment with bromodimethylborane. Radioactively labeled fatty acid ethanolamides were prepared by the reaction of acid chlorides (Nu-Check

Prep) with [^HJethanolamine (10-30 Ci/mmol; American Radiolabeled Chemicals) as described by Desarnaud (1995) J. Biol. Chem. 270:6030-6035. All compounds were o purified by high-performance liquid chromatography or flash column chromatography and their identities were established by nuclear magnetic resonance and/or gas chromatography-mass spectrometry. Additional compounds were purchased from Avanti Polar Lipids, Cayman Chemical Co., Nu-Check Prep, RBI or Sigma.

One exemplary test to screen for compounds useful in the methods of 5 the invention is the [^H] Anandamide Competition Assay, which uses tissue culture cells. Human CCF-STTG1 astrocytoma cells (American Type Culture Collection) were grown in RPMI 1640 culture medium containing 10% fetal bovine serum and 1 mM glutamine. For standard competition assays, confluent cells grown in 24-well plates were rinsed and pre-incubated for 10 min at 37°C in Tris-Krebs' buffer (NaCI, 0 136 mM; KC1, 5 mM; MgCl2-6H2θ, 1.2. mM; CaCl2-2H2θ, 2.5. mM; glucose, 10 mM; Trizmabase, 20 mM) containing 0.1-0.3% dimethylsulfoxide (DMSO) or 0.1- 0.3% DMSO plus test compounds at their final concentrations (0.1-100 μM). After having discarded the pre-incubation media, the cells were incubated for 4 min in 0.4 ml of Tris-Krebs' buffer containing 30 nM [^H] anandamide (220 Ci/mmol, New 5 England Nuclear) and 0.1-0.3% DMSO, or 0.1-0.3% DMSO plus test compounds. Reactions were stopped by removing the incubation media and rinsing the cells 3 times with 0.4 ml of ice-cold Tris-Krebs' buffer containing 0.1% fatty acid-free bovine serum albumin (Sigma). Radioactive material in Triton X-100 cell extracts was measured by liquid scintillation counting. Preliminary analyses carried out by 0 thin-layer chromatography demonstrated that >95% of this radioactive material was non-metabolized H]anandamide, suggesting that our astrocytoma cell preparation contains no significant anandamide amidohydrolase activity.

Another exemplary test to screen for compounds useful in the methods of the invention is the [^H] Anandamide Transport Assay. For standard transport assays, confluent astrocytoma cells grown in 90-mm plates were incubated at 37°C in

10 ml of Tris-Krebs' buffer containing 10-50x10^ dpm/ml of one the following radioactive tracers (unless indicated otherwise, specific radioactivity was 0.31-0.69 mCi/mmol): [^H] anandamide (220 Ci/mmol, New England Nuclear),

[3H]oleylethanolamide(18:lΔ9), [^Hjeicosaenoyl-ethanolamide (20:lΔl 1), [^HJeicosadienoylethanolamide (20:2ΔH>14)₅ [3H]eicosa-trienoylethanolamide

(20:3Δ⁸»¹ !>¹⁴), [ H]2-arachidonylglycerol (100 mCi/mmol; New England Nuclear, custom-synthesized), [3H]N-(4-hydroxyρhenyι) arachidonamide (200 Ci/mmol; American Radiolabeled Chemicals). At various times after the addition of tracer (0-20 min), 1-ml samples of the incubation media were collected for liquid scintillation counting.

Another exemplary test to screen for compounds useful in the methods of the invention is to measure transport kinetics. Cells were incubated for 4 min at

37°C in the presence of 10 to 500 nM anandamide containing 0.05 to 2.5 nM

[³H]anandamide. Non-specific accumulation (measured at 0-4°C) was subtracted before determining kinetic constants by Lineweaver-Burke analysis.

To analyze data, a minimum of three independent experiments conducted in triplicate was used to define the concentration needed to produce half- maximal inhibition (IC50) for each compound. IC50 values obtained by non-linear least square fitting of the data were converted to Ki values by the Cheng-Prusoff equation (see, e.g., Cheng (1973) Biochem. Pharmacol. 22-23:3099-3108) using the apparent Michaelis constant (Km) determined from kinetic experiments (0.6 μM). All other experiments were carried out in triplicate, and repeated at least twice with identical results. Data are expressed as mean + s.e.m. Molecular modeling was conducted on an SGI Octane R10000 workstation with the Tripos Sybyl 6.4™ and Tripos™ empirical force field molecular modeling package. The initial structures were generated using standard bond lengths and angles from the Sybyl™ package. Charges were calculated for all molecules by the semi-empirical method (MOP A/AMI), and energies minimized using the Tripos force field in two stages. First, the steepest descent method was applied for the first 200 steps, followed by the conjugate gradient method until the maximum derivative was less than 0.001 kcal/mole/A. The conformation of anandamide was refined using the Tripos random search module on the six rotatable single bonds within the group of four non- conjugated cis double bonds. Conformer 1 (depicted in Figure 3) has a hairpin conformation similar to that described by others for anandamide (Barnett-Norris (1997) in 1997 Symposium on the Cannabinoids, International Cannabinoid Research Society, p. 5) and arachidonic acid (Rich (1993) Biochim. Biophys. Acta 1178:87-96). The preferred conformers of all anandamide analogs were generated from conformer 1 by appropriate structural modifications followed by minimization.

Screening by measuring ft H] Anandamide transport in astrocytoma cells

As expected of a carrier-mediated process, [^HJanandamide accumulation in human astrocytoma cells is rapid (t 1/2=3 min), temperature- dependent and saturable, displaying an apparent Michaelis constant (Km) of 0.6 μM 5 and a maximal accumulation rate (Nmax) of 14.7 pmol/min/mg of protein. The accumulation is not affected by replacement of Νa⁺ with choline, suggesting that is mediated by a Na⁺-independent mechanism. In addition, [^Hjanandamide accumulation is prevented by the anandamide transport inhibitor N-(4- hydroxyphenyl)-arachidonamide (AM404, 22 in Fig. 2) with a K, value of 2.1+0.2 o μM, whereas its positional isomer N-(3-hydroxyphenyl)-arachidonamide (23 in Fig. 2) is 10 times less effective. A variety of compounds that are substrates or inhibitors of membrane transporters (including prostaglandins, leukotrienes, organic anions, ceramide, urea, amino acids and biogenic amines) have no effect on [^HJanandamide accumulation when tested at concentrations ranging from 10 to 100 μM. Together, 5 these results indicate that [³H] -anandamide accumulation in human astrocytoma cells is mediated by a high-affinity, Νa⁺-independent transporter functionally analogous to that described in rat brain neurons and astrocytes (Beltramo (1997) Science 277:1094- 1097).

Screening by competition with ft H] anandamide transport

To

of substrates and inhibitors with the anandamide transporter, a series of anandamide analogs was synthesized and examined for their abilities to interfere with

[^H] anandamide uptake. The anandamide structure reveals three potential pharmacophores which lend themselves to structural modification: (A) the highly hydrophobic cts-tetraene carbon chain; (B) the polar carboxamido group; and (C) the hydroxyethyl head group. The correlation between ligand structure and function can be determined by systematically varying the structures of these three components. For the A pharmacophore, results indicate that analogs incorporating either a C- 18 or a C-20 hydrophobic tail with one, two or three non-conjugated cis double bonds in the middle part of the chain (Figure 1; 2-6) compete successfully with

[^Hjanandamide for transport. By contrast, analogs with fully saturated chains or with a trans or terminal double bond fail to do so (Figure 1; 7-10).

Exploration of the B pharmacophore reveals that compounds containing primary (Fig. 2; 12), secondary (Fig. 2; 11) and tertiary carboxamido groups (e.g., Figure 3; 28-31) as well as hydroxyethyl ester (Fig. 2; 13) or glycerol ester moieties (Fig. 2; 18, 19) are capable of competing with [-1H] anandamide, but exhibit a wide range of potencies. Conversely, compounds containing a free carboxylic acid (Fig. 2; 16), carboxyethyl or carboxymethyl groups (Fig. 2; 14, 15), or a primary alcohol (Fig. 2; 17) are inactive.

Structural variations of the hydroxyethyl head group (C) also lead to compounds with diverse selectivities for the anandamide transporter. Thus substitution of the terminal hydroxy with a methyl group (Fig. 2; 11) causes a substantial decrease in potency, while elimination of the hydroxyethyl moiety yields compounds that are as potent as anandamide, as illustrated by arachidonamide (Fig. 2; 12) or oleamide Kj = 11.1+2.6 μM; not shown). Introduction of a methyl group alpha to the amido nitrogen also leads to active compounds (Fig. 2; 20, 21). These chiral molecules display considerable enantioselective inhibition of [^HJanandamide transport, hi the 1 -methyl series illustrated in Fig.2, the (S) enantiomer 21 is approximately 4 times more potent than its (R) isomer 20. Similar enantioselectivity was also demonstrated with an additional series of analogs in which the methyl group was introduced beta to the amido nitrogen. The enantioselectivity for anandamide transport displayed by 20 and 21 is congruent to that demonstrated for anandamide amidohydrolase, but opposite to that for the CB1 receptor (Abadjj (1994) supra).

To screen for the effects of head group conformational preference, a set of analogs was synthesized in which the hydroxyalkyl group is partially restricted by incorporation into five- or six-membered rings. The resulting 3- and 4- hydroxypiperidinyl- (Fig. 2; 29 and 30) and 3-hydroxypyrrolidinyl- (Fig. 2; 31) amides have biological activities similar to that of anandamide. Of these, 29 is optically active while 30 and 31 are racemic pairs. Another cyclic head group analog (Fig. 2; 28) has both amido nitrogen and hydroxyl oxygen restricted into a morpholine ring. This analog maintains considerable activity, approximately one half that of anandamide, indicating that the hydrogen in the hydroxyl head group may not be necessary to interact with the transporter.

The most striking structure-activity correlation was observed with analogs having phenyl substitutions at the head group. Replacement of the hydroxyethyl with a hydroxyphenyl group leads to relatively potent uptake inhibitors, with the 4-hydroxyphenyl analog (AM404, 22, Fig. 2) being distinctly the most successful. Conversely, the 4-methylphenyl analog 25 (Fig. 2) as well as other analogs with electron-donating (Fig. 2; 24) or electron-withdrawing (Fig. 2; 26, 27) para substituents display no significant activity. Varying these substituents from the para to the meta or ortho position does not restore activity. Other analogs containing multiple substituents on the phenyl ring, e.g., 3-chloro-4-hydroxyphenyl, or a bulkier aromatic moiety, e.g., l-(4-hydroxynaphthyl), are also less potent than 22.

Competition experiments are also very useful for screening for compounds useful in the methods of the invention. Competition experiments outline the structural requirements for ligand recognition by the anandamide transporter, but do not provide information on whether the ligands may also serve as substrates for the transporter. To investigate substrate translocation a representative set of radioactively labeled compounds is used. Three key analogs that compete with anandamide for uptake are used: [³H] -arachidonamide (Fig. 2; 12), [³H]N-(4- hydroxyphenyl)arachidonamide (A404) (Fig. 2; 22), the most potent competitor in our series, and [³H]2-arachidonylglycerol (Fig. 2; 18), an endogenous cannabimimetic substance produced in brain hippocampus during neuronal activity. The three analogs are transported as rapidly and effectively as [³H]anandamide. These findings suggest that the anandamide transporter may also participate in the inactivation of 2- arachidonylglycerol, which was thought to be primarily mediated by enzymatic hydrolysis (Goparaju (1998) FEBS Lett. 422:69-73; Schaeffer (1994) Cell 79:427- 436). In agreement with this possibility, kinetic analyses indicate that [³H]2- arachidonylglycerol is accumulated in astrocytoma cells with an apparent K_m of 0.7 μM and a Nmax of 28 pmol/min mg protein, values that are comparable to those obtained with [³H]-anandamide in the same cell preparation. The effects of modifications in the hydrophobic tail are also useful in the screening of compounds useful in the methods of the invention. [³H] anandamide and one cw-triene analog ([³H]eicosatrienoylethanolamide, 20:3Δ8>H,14)₅ one cis- diene analog ([³H] eicosadienoylethanolamide, 20:2ΔH'14)₅ and two cis monounsaturated analogs with the double bond located in the middle of the carbon chain (oleylethanolamide, [³H]18:1Δ9; and eicosaenoylethanolamide, [³H]20:lΔl ) are used in this screening assay. Although all of these fatty acid ethanolamides are able to compete with [ H] anandamide for transport, only [ H] anandamide is effectively transported into cells. Of the remaining compounds, the et-j-triene and the czs-diene are transported very slowly (t 20 min), while the two monoalkenes are not transported at all. [ HjPalmitylethanolamide (16:0) is not transported to any significant extent. These results reveal the existence of two distinct sets of structural requirements in the function of the anandamide transporter; one for substrate recognition and another for substrate translocation.

These results provide guidelines on the structural requisites of substrates and inhibitors of the anandamide transporter that can be used to design and screen for compounds useful in the methods of the invention: (1) The stereoselectivity of these requirements is illustrated by the finding that S-l'-methylanandamide (Fig. 2; 21) is significantly more potent than its R-isomer (Fig. 2; 20) at competing with [³H] anandamide for transport, and is further supported by similar results obtained with chiral 2'-methylanandamides. The configurational preference of 1 '-methylanandamides is opposite to that found at the CB1 cannabinoid receptor and may prove useful when designing transport inhibitors devoid of direct CB1 receptor activity.

(2) A polar non-ionizable head group is of primary importance for a productive interaction with the anandamide transporter. Although this interaction may be enhanced by a hydrogen-donating hydroxyl group, polar groups with hydrogen- acceptors may also yield relatively potent compounds (see, e.g., the ether-containing analog 28; Fig. 2). The results obtained with phenolic amides, such as N-(4- hydroxyphenyl)-arachidonamide (22; Fig. 2) and its meta analog 23; Fig. 2, further indicate that a hydroxy moiety strongly favors the interaction with the transporter, and underscore the stringent regiochemical requirements of such interaction.

(3) Although the presence of a secondary amide moiety in the polar carboxamido group may improve affinity for the transporter, compounds that contain an ester bond are also effective, provided that an appropriately spaced hydroxyl group is also present (e.g., 13 and 14; Fig. 2). An interesting corollary of this property is that the anandamide transporter may participate in the biological inactivation of both anandamide and 2-arachidonylglycerol, a possibility supported by the similar transport kinetics of these two substrates.

(4) Modifications of the hydrophobic fatty acid tail reveal unexpectedly distinct requirements for recognition and translocation of substrates by the anandamide transporter. Substrate recognition requires the presence of at least one cis double bond situated at the middle of the fatty acid chain, pointing to a preference for ligands in which the hydrophobic tail can fold in the middle and adopt a bent U-shaped conformation. Indeed, analogs with fully saturated chains or those incorporating trans double bonds do not interact significantly with the transporter. By contrast, substrate translocation requires a minimum of four cis non-conjugated double bonds, as ligands containing one, two or three olefins are transported either very slowly (2, 3; Fig. 1) or not at all (4, 6; Fig. 1). This suggests that for transmembrane transport to occur substrates must be capable of adopting a tightly folded conformation, one that is not energetically favorable for ligands containing an insufficient number of cis double bonds.

Molecular modeling studies of fatty acid ethanolamides differing in the degree of unsaturation of their hydrophobic carbon chains provides insight into these distinctive conformational requirements. Low-energy conformers of these molecules are significantly different. The presence of one or more non-conjugated cis double bonds in the middle of the chain leads to the formation of a turn that brings in closer proximity head and tail of the molecule. The shape of this turn is determined by the number and position of the cis double bonds. Conversely, the introduction of a central trans double bond yields a more extended chain conformation, and hinders the ability of the molecule to undergo folding. Thus one of the low-energy conformers of anandamide displays a folded hairpin shape with the two halves of the molecule facing each other. The 6 c-s-triene analog may adopt an analogous conformation, though one that is considerably wider than that of anandamide. The width of the turn increases considerably in the two cts-dienes and the two monoalkenes, as illustrated by the marked increase in distance between head group and tail of the molecule, yielding a series of cognate U-shaped conformers. Importantly, while anandamide may adopt either a closed-hairpin or a U-shaped conformation depending on the properties of the surrounding milieu, like its parent molecule arachidonic acid, the hairpin conformation may be thermodynamically unfavorable to fatty acid ethanolamides containing only one or two double bonds. Thus, the initial recognition step may require that substrates assume a bent U-shaped conformation of variable width. Subsequent steps of translocation across the cell membrane may impose a more tightly folded hairpin conformation.

In summary, these exemplary techniques and guidelines provide clear parameters to select for compounds useful as pharmaceutical compositions in the methods of the invention. Endogenous cannabinoids The methods of the invention comprise administration of pharmaceutical compositions able to inhibit the inactivating transport of an endogenous cannabinoid, such as, e.g., anandamide or 2-arachidonylglycerol (2-AG). Anandamide, or arachidonylethanolamide, is an endogenous derivative of arachidonic acid that binds with high affinity to cannabinoid receptors and mimics virtually all pharmacological actions of plant-derived or synthetic cannabinoid drugs (Devane (1992) Science 258:1946-1949). In vivo, anandamide may be produced physiologically tlirough enzymatic cleavage of the phospholipid precursor, N- arachidonyl phosphatidylethanolamine (Di Marzo (1994) Nature 372: 686-691; Cadas (1996) J. Neurosci. 16: 3934-3942; Sugiura (1996) Eur. J. Biochem. 240: 53-62; Cadas (1997) J. Neurosci. 17: 1226-1242), a reaction that may be triggered by the stimulation of neurotransmitter receptors (Di Marzo (1994) supra; Giuffrida (1999) Nature Neurosci. 2: 358-363. After release, anandamide is disposed of through a rapid inactivation process consisting of uptake into cells (Beltramo (1997) Science 277: 1094-1097; Hillard (1997) J. Neurochem. 69: 631-638), followed by catalytic hydrolysis (Desarnaud (1995) J. Biol. Chem. 270: 6030-6035; Ueda (1995) J. Biol. Chem. 270: 23823-23827; Cravatt (1996) Nature 384: 83-87. Anandamide uptake is a Na⁺-independent process that fulfills four key criteria that define carried-mediated transport: high affinity, temperature dependence, substrate selectivity and substrate saturation (Beltramo (1997) supra; Hillard (1997) supra.

Homo- -linolenyl ethanolamide and docosatetraenyl ethanolamide are additional naturally occurring cannabinoids that bind to cannabinoid receptors, see, e.g., Deutsch (1997) NTDA Res Monogr. 173:65-84.

2-arachidonoylglycerol is a multifunctional lipid mediator in the nervous and immune systems, see, e.g., Sugiura (2000) Ann. NY Acad. Sci. 905:344- 346; Sugiura (2000) Biochem. Biophys. Res. Commun. 271:654-658.

Neuropsychiatric Disorders The invention provides methods for ameliorating neuropsychiatric disorders. In one embodiment, inhibiting the inactivating transport of an endogenous cannabinoid substance counteracts the effects of dopamine hyperactivity. In alternative embodiments, the neuropsychiatric disorders include schizophrenia, schizo affective disorder, schizophreniform disorder, borderline personality disorder, attention-deficit hyperactivity disorder, autism spectrum disorder, Tourette's syndrome or a psychoactive substance-induced organic mental disorder or a psychoactive substance use disorder. Any of the many in vitro or in vivo art-accepted assays or animal models for treating neuropsychiatric disorders can be used to demonstrate that a pharmaceutical composition effectively ameliorates a neuropsychiatric disorder; e.g., by counteracts the effects of dopamine hyperactivity. For example, one well-known model tests the ability of a compound to antagonize the hyperactivity caused by dopamine infusion into the nucleus accumbens of a rat; see, e.g., U.S. Patent No. 4,877,794.

Schizophrenia is a common and serious neuropsychiatric disorder characterized by loss of contact with reality (psychosis), hallucinations (false perceptions), delusions (false beliefs), abnormal thinking, flattened affect (restricted range of emotions), diminished motivation, and disturbed work and social functioning. For a method for diagnosing and testing for the ability to ameliorate schizophrenia, see, e.g., U.S. Patent Nos. 6,051,605; 5,837,730.

Schizoaffective disorder is a neuropsychiatric "psychotic" disorder characterized by significant mood symptoms (depression or mania) and symptoms of schizophrenia. The diagnosis requires that mood symptoms be present for a substantial portion of the total duration of illness. Differentiating schizoaffective disorder from schizophrenia and affective disorder may require longitudinal assessment of symptoms and symptom progression. The prognosis is somewhat better than that for schizophrenia but worse than that for mood disorders. For a method for diagnosing and testing for the ability to ameliorate schizoaffective disorder, see, e.g., U.S. Patent Nos. 5,663,167; 5,869,490; 5,627,178.

Schizophreniform disorder is a neuropsychiatric disorder with symptoms that are identical to those of schizophrenia but last 1 to 6 months. At presentation, the diagnosis is usually unclear. Psychosis secondary to substance abuse or to a physical disorder must be ruled out. Persistence of symptoms or disability beyond 6 months suggests schizophrenia, but the acute psychosis may also evolve into a psychotic mood disorder, such as bipolar or schizoaffective disorder. Longitudinal observation is often required to establish the diagnosis and appropriate treatment. For a method for diagnosing and testing for the ability to ameliorate schizophreniform disorder, see, e.g., U.S. Patent No. 5,736,541; 5,663,167; 5,627,178. Tourette's syndrome is a neuropsychiatric disorder more prevalent in males than in females. The movement disorder may begin with simple tics that progress to multiple complex tics, including respiratory and vocal ones. Nocal tics may begin as grunting or barking noises and evolve into compulsive utterances. Coprolalia (involuntary scatologic utterances) occurs in 50% of patients. Tics tend to be more complex than myoclonus, but less flowing than choreic movements, from which they must be differentiated. The patient may voluntarily suppress them for seconds or minutes. For a method for diagnosing and testing for the ability to ameliorate Tourette's Syndrome, see, e.g., U.S. Patent No. 6,075,028, describing a method for treating same.

Autism spectrum disorder is a neuropsychiatric disorder of early childhood characterized by abnormal social relationships; language disorder with impaired understanding, echolalia, and pronominal reversal, rituals and compulsive phenomena (as an insistence on the preservation of sameness) and uneven intellectual development with mental retardation in most cases. Autism is two to four times more common in boys than girls. The concordance rate is significantly greater in monozygotic than dizygotic twins, indicating the importance of genetic factors. The syndrome is defined by its behavioral manifestations. The level of intellectual function and the presence or absence of neurologic damage are recorded separately using a multiaxial diagnostic system. CT scans have isolated a subgroup of autistic children with enlarged ventricles. MRI has identified a subgroup of autistic adults with hypoplasia of the cerebellar vermis. Individual cases of autism have been associated with the congenital rubella syndrome, cytomegalic inclusion disease, phenylketonuria, and the fragile X syndrome. For a method for diagnosing and testing for the ability to ameliorate autism spectrum disorder, see, e.g., U.S. Patent No. 6,020,310.

Attention-deficit hyperactivity disorder is a neuropsychiatric disorder having a persistent and frequent pattern of developmentally inappropriate inattention and impulsivity, with or without hyperactivity. This definition of attention deficit disorder (ADD), from the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-TV), shifts the focus from excessive physical activity. ADD is implicated in learning disorders and can influence the behavior of children at any cognitive level, except for moderate to profound mental retardation. ADD affects about 5 to 10% of school-aged children, accounting for half of the childhood referrals to diagnostic clinics. ADD tends to occur in families and is common in first-degree biological relatives. ADD with hyperactivity and impulsivity is seen 10 times more frequently in boys than girls. Many now believe that ADD is a difference rather than a deficit or disorder in brain biochemistry resulting in a difference in approach to learning. For a method for diagnosing and testing for the ability to ameliorate attention-deficit hyperactivity disorder, see, e.g., U.S. Patent Nos. 6,034,101; 5,885,998; 5,874,090. Borderline personality disorder is a neuropsychiatric disorder that, as described in Has Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV), can be divided into three types of personality disorders: A) odd/eccentric, B) dramatic/erratic, and C) anxious/inhibited. For a method for diagnosing and testing for the ability to ameliorate borderline personality disorder, see, e.g., U.S. Patent No. 5,985,322; 5,910,319; 5,627,196; 5,589,512.

Animal Models for Confirming In Vivo Efficacy

In alternative embodiments, the neuropsychiatric disorders ameliorated by the methods of the invention include those at least in part caused or mediated by dopamine hyperactivity, including, e.g., schizoaffective disorder, schizophreniform disorder, borderline personality disorder, attention-deficit hyperactivity disorder, autism spectrum disorder, Tourette's syndrome or a psychoactive substance-induced organic mental disorder or a psychoactive substance use disorder. Art-accepted animals models for these disorders are used to confirm the in vivo efficacy of the pharmaceutical composition inhibitors of endogenous cannabinoid substance inactivating transport. For example, the methods of the invention have been demonstrated to ameliorate neuropsychiatric disorders by determining the behavioral effects of AM404 using art-recognized animal models, as described in the Examples, below. These animal (rat) models are predictive of antipsychotic activity; and include (1) the motor hyperactivity induced by dopamine agonists quinpirole in rats, and (2) the yawning response induced by the dopamine agonist apomorphine in rats. For the animal models for studying autism, see, e.g., Ingram (2000) Neurotoxicol Teratol. 22:319-324, describing that prenatal exposure of rats to valproic acid reproduces the cerebellar anomalies associated with autism. For the animal models for studying schizophrenia, see, e.g., Swerdlow (2000) J. Neurosci. 20:4325-4336, describing that sensorimotor gating, measured by prepulse inhibition (PPI) of the startle reflex, is reduced in schizophrenia patients and in rats treated with dopamine agonists. An animal model for psychosis is, e.g., the behavior induced by the N-methyl-D-aspartate receptor antagonist dizocilpine maleate in rats, as described by, e.g., Andine (1999) J. Pharmacol. Exp. Ther. 290:1393-1408.

Treating Neuropsychiatric Disorders Using the Methods of the Invention

The invention provides methods for ameliorating various neuropsychiatric disorders by administering a pharmaceutical composition able to inhibit the inactivating transport of an endogenous cannabinoid. The pharmaceutical compositions used in the methods of the invention can be administered by any means known in the art, e.g., parenterally, topically, orally, or by local administration, such as by aerosol or transdermally. The pharmaceutical compositions can be formulated in any way and can be admimstered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA ("Remington's").

Pharmaceutical formulations can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such drugs can contain sweetening agents, flavoring agents, coloring agents and preserving agents. A formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture.

Pharmaceutical formulations for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained through combination of inhibitors of inactivating transport compounds with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores. Suitable solid excipients are carbohydrate or protein fillers include, e.g., sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropyhnethyl-cellulose, or sodium carboxy-methylcellulose; and gums including arabic and tragacanth; and proteins, e.g., gelatin and collagen. Disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound (i.e., dosage). Pharmaceutical preparations of the invention can also be used orally using, e.g., push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules can contain active agents mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers, hi soft capsules, the active agents can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.

Aqueous suspensions can contain an active agent (e.g., N-(4- hydroxyphenyl) arachidonamide) in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin. Formulations can be adjusted for osmolarity.

Oil-based pharmaceuticals are particularly useful for administration of hydrophobic active agents. Oil-based suspensions can be formulated by suspending an active agent (e.g., N-(4-hydroxyphenyl) arachidonamide) in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. See e.g., U.S. Patent No. 5,716,928 describing using essential oils or essential oil components for increasing bioavailability and reducing inter- and infra-individual variability of orally administered hydrophobic pharmaceutical compounds (see also U.S. Patent No. 5,858,401). The oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid. As an example of an injectable oil vehicle, see Minto (1997) J. Pharmacol. Exp. Ther. 281:93-102. The pharmaceutical formulations of the invention can also be in the form of oil-in- water emulsions. The oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono- oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water can be formulated in admixture with a dispersing, suspending and/or wetting agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients, e.g., sweetening, flavoring and coloring agents, can also be present.

In the methods of the invention, the pharmaceutical compounds can also be administered by in intranasal, intraocular and intravaginal routes including suppositories, insufflation, powders and aerosol formulations (for examples of steroid inhalants, see Rohatagi (1995) J. Clin. Pharmacol. 35:1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol. 75:107-111). Suppositories formulations can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug. Such materials are cocoa butter and polyethylene glycols.

In the methods of the invention, the pharmaceutical compounds can be delivered by transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. In the methods of the invention, the pharmaceutical compounds can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug which slowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed. 7:623-645; as biodegradable and injectable gel formulations, see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oral administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674 . Both transdermal and intradermal routes afford constant delivery for weeks or months.

In the methods of the invention, the pharmaceutical compounds can be provided as a salt and can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder in 1 mM to 50 mM histidine, 0.1% to 2% sucrose, 2% to 7% mannitol at a pH range of 4.5 to 5.5, that is combined with buffer prior to use. In the methods of the invention, the pharmaceutical compounds can be parenterally administered, such as by intravenous (IN) administration or administration into a body cavity or lumen of an organ. These formulations will commonly comprise a solution of active agent dissolved in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter. These formulations may be sterilized by conventional, well known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. For IN administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a suspension in a nontoxic parenterally-acceptable diluent or solvent, such as a solution of 1,3-butanediol.

In another embodiment, the formulations of the invention can be delivered by the use of Hposomes which fuse with the cellular membrane or are endocytosed, e.g., by employing ligands attached to the liposome, or attached directly to the oligonucleotide, that bind to surface membrane protein receptors of the cell resulting in endocytosis. By using Hposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent into target cells in vivo. See, e.g., U.S. Patent Νos. 6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13:293-306; Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46:1576-1587.

In the methods of the invention, a pharmaceutical composition is administered in an amount sufficient to inhibit the inactivating transport of an endogenous cannabinoid substance and to ameliorate a neuropsychiatric disorder. The amount of pharmaceutical composition adequate to accomplish this is defined as a "therapeutically effective dose." The dosage schedule and amounts effective for this use, i.e., the "dosing regimen," will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age and the like, h calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.

The dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108; the latest Remington's, supra). The state of the art allows the clinician to determine the dosage regimen for each individual patient, active agent and disease or condition treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance to determine the dosage regiment, i.e., dose schedule and dosage levels, of any inhibitor of endogenous cannabinoid cell uptake administered practicing the methods of the invention.

Single or multiple administrations of formulations can be given depending on the dosage and frequency as required and tolerated by the patient. The formulations should provide a sufficient quantity of active agent to effectively treat the neuropsychiatric disorder. For example, one typical pharmaceutical formulations for oral administration of N-(4-hydroxyphenyl) arachidonamide (AM404) is in a daily amount of between about 0.5 to about 20 mg per kilogram of body weight per day. In an alternative embodiment, dosages are from about 1 mg to about 4 mg per kg of body weight per patient per day are used. Lower dosages can be used, particularly when the drug is administered to an anatomically secluded site, such as the cerebral spinal fluid (CSF) space, in contrast to administration orally, into the blood stream, into a body cavity or into a lumen of an organ. Substantially higher dosages can be used in topical administration. Actual methods for preparing parenterally adminisfrable formulations will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's, supra. See also Nieman, In "Receptor Mediated Antisteroid Action," Agarwal, et al., eds., De Gruyter, New York (1987).

Kits

The invention provides kits that contain pharmaceutical compositions and instructions specifically useful in practicing the methods of the invention. After a pharmaceutical comprising an inhibitor of an inactivating transport of an endogenous cannabinoid substance has been formulated in a acceptable carrier, it can be placed in an appropriate container and labeled for treatment of an indicated condition, e.g., a neuropsychiatric disorder. Labeling would include, e.g., instructions concerning the amount, frequency and method of administration. In one embodiment, the invention provides for a kit for the treatment of a neuropsychiatric disorder in a human or other animal which includes an inhibitor of an inactivating transport of an endogenous cannabinoid substance and instructional material teaching the indications, dosage and schedule of administration of the inhibitor.

Kits containing pharmaceutical preparations (e.g., vectors, nucleic acids) can include directions as to indications, dosages, routes and methods of administration, and the like. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

EXAMPLES

The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1: AM 404 Effectively Inhibits Behaviors Predictive of Antipsychotic Activity In Vivo

The following example demonstrates that the methods of the invention provide an improved and efficacious means to ameliorate neuropsychiatric disorders.

This results discussed in this example demonstrate that inhibitors of the inactivating transport of an endogenous cannabinoid can ameliorate neuropsychiatric disorders. This was determined by measuring the behavioral effects of one of the exemplary compounds that can be used in the methods of the invention, AM404. AM404 is a selective inhibitor of endogenous cannabinoid transport. Two art-recognized animal models were used that are predictive of antipsychotic activity: (1) the motor hyperactivity induced by dopamine agonists (quinpirole, SKF-812g7) in rats, and (2) the yawning response induced by the dopamine agonist apomorphine in rats. In both models, AM404 produced a highly effective inhibition of dopamine agonist-induced hyperactivity, when injected at intraperitoneal doses of 10 to 20 mg/kg, or intra- cerebroventricular doses of 2 to 10 μg/rat. Importantly AM404 had little effect when administered alone, indicating that its actions are selectively expressed during dopamine hyperactivity, a feature that is highly desirable therapeutically.

Cannabinoid receptors, the target of the marijuana constituent ⁹- tetrahydrocannabinol, are densely expressed in basal ganglia and cortex, regions of the central nervous system (CNS) that are critical for the control of cognition, motivation and movement (see, e.g., Herkenham (1990) Proc. Natl. Acad. Sci. USA 87:1932-1936; Matsuda (1993) J. Comp. Neurol. 327:535-550; Tsou (1998)

Neuroscience 83:393-411). This distribution provides multiple opportunities for functional interactions between endogenous cannabinoid substances, such as anandamide (Devane et al., 1992) Science 258:1946-1949; Di Marzo (1994) supra), and ascending dopamine pathways. That these interactions may occur in vivo is indicated by several observations. First, in the striatum of freely moving rats anandamide release is greatly increased after activation of D₂-family receptors with the selective agonist quinpirole (Giuffrida (1999) Nature Neurosci. 2:358-363). Second, pretreatment with the CBl cannabinoid antagonist SR141716A enhances the stimulation of motor behavior elicited by systemic administration of quinpirole (Giuffrida (1999) supra), though it has little effect per se on basal motor activity (Rinaldi-Carmona (1994) FEBS Lett. 350:240-244); Compton (1996) J. Pharmacol. Exp. Ther. 277:586-594); Navarro (1997) NeuroReport 8:491-496). Third, injection of D₂-family agonists into basal ganglia nuclei opposes the behavioral response to locally administered CBl receptor agonists (Sanudo-Pena (1996) Neurosci. Lett. 206:21-24); Safiudo-Peήa (1998) Synapse 30:221-226). Finally, chronic treatment with D₂-family antagonists results in upregulated expression of CBl receptor mRNA in striatum(Mailleux (1993) J. Neurochem. 61:1705-1712). Together, these findings suggest that one of the functions of anandaniide in the CNS may be to modulate dopamine D₂-receptor induced facilitation of psychomotor activity. In agreement with this possibility, anandamide and other CBl agonists inhibit movement, produce catalepsy, and attenuate d-amphetamine-induced hyperactivity and stereotypy (Gorriti (1999) Eur. J. Pharmacol. 365:133-142), while disruption of the CBl receptor gene profoundly affects movement control (Ledent (1999) Science 283:401-404); Zimmer (1999) Proc. Natl. Acad. Sci. USA 96:5780-5785).

When anandamide is administered as a drug, its effects are curtailed by a two- step mechanism consisting of transport into cells, mediated by a high-affinity carrier system (Beltramo (1997) Science 277:1094-1097; Hillard (1997) J. Neurochem.

69:631-638; Piomelli (1999) supra), followed by intracellular hydrolysis, catalyzed by a relatively non-selective amidohydrolase enzyme (Deutsch (1993) Biochem. Pharmacol. 46:791-796). Consequently, the anandamide transport inhibitor, AM404 [N-(4-hydroxyphenyl)-arachidonamide], prolongs and enhances several responses to exogenous anandamide, including analgesia (Beltramo (1997) Science 277: 1094- 1097) and vasodilatation (Calignano (1997) Eur. J. Pharmacol. 337:R1-R2).

The invention shows that blockade of anandamide transport, by causing this lipid to accumulate at its sites of release, can control aspects of dopamine neurotransmission and offers a pharmacological strategy to correct pathological conditions characterized by dopaminergic dysfunction. To demonstrate this hypothesis, potential sites of action and pharmacological properties of AM404 in the CNS were identified. The effects of this drug on behavioral responses elicited by the activation of D -family receptors was also demonstrated.

Characterization of anandamide transport. Coronal slices (0.45 mm thick) from adult rat brain were prepared with a vibratome and split along the midline

5 with a razor blade. Each half was collected separately and allowed to equilibrate for 2.5 h at 37 °C in Tris-Krebs' buffer (NaCI 136 mM, KC15 mM, MgCl₂ 1.2 mM, CaCl₂2.5 mM, glucose 10 mM, Trizma base 20 mM; pH 7.4) aerated with 5% CO₂ in O . The slices were incubated under agitation for 10 min in Tris-Krebs' buffer containing test compounds, followed by a 5-min incubation in the presence of o [³H]anandamide (30 nM, 1.810⁵ dpm/ml; 221 Ci/mmol, New England Nuclear,

Wilmington, DE) and appropriate concentrations of vehicle or test compounds. In all experiments SR141716A (0.1 M; RBI, Natick, MS, as part of the Chemical Synthesis Program of the NTMH (NO1MH30003)) was added to the incubations to prevent binding of [³H] anandamide to CBl receptors. At the end of the incubation 5 period, the slices were rinsed with Tris-Krebs' buffer containing fatty acid-free bovine serum albumin (BSA, 0.1%), homogenized in Tris-Krebs' buffer/methanol (1:1, v/v), and radioactivity was measured by liquid scintillation counting.

Selectivity ofAM404. [³H]AM404 (arachidonyl-5,6,8,11,12,14,15-³H; 200-240 Ci/mmol; American Radiolabeled Chemicals Inc., St. Louis, MO) was 0 radioactively pure (>99%) by high performance liquid chromatography. [ HJAM404 (10 g containing 2.8xl0⁶ dpm in 5 1 of DMSO) was administered by intracerebro ventricular (icv) injection in cannulated rats (Taconic Farms, Germantown, NY) through a calibrated polyethylene- 10 tubing. Average recovery of [³H]AM404 after passage through the tubing was 37.6+2.5 % (n = 4). Animals were 5 sacrificed 3 min after injections and individual brain regions were dissected, weighed and homogenized. Radioactivity in homogenates was measured by liquid scintillation counting. Identical distributions of radioactivity were obtained when measurements were carried out 60 min after injection of [³H]AM404. AM404 had low affinity (concentration needed to produce half-maximal response, EC₅0 >10 M) for the 0 following targets. Receptors: adenosine A._\ (rat brain, [³H]DPCPX), alphai adrenergic non-selective (rat brain, [³H]prazosin), alpha₂ adrenergic non-selective (rat brain cortex, [³H]rauwolscine), alphai adrenergic (human, [¹²⁵I]cyanopindolol), alpha₂ adrenergic (human, [³H]CGP-12117), Oι dopamine (human recombinant, [³H]SCH23390), D_2L dopamine (human recombinant, [³H]spiρerone), 5-HTι serotonin (rat brain cortex, [³H] serotonin), 5-HT serotonin (rat brain, [³H]ketanserin), M₂ muscarinic (human recombinant, [ H]NMS), M₃ muscarinic (human recombinant, [³H]NMS), delta-opioid (guinea pig brain, [³H]DPDPE), kappa-opioid (guinea pig brain, [³H]U-69593), -opioid (guinea pig brain, [³H]DAMGO), non-selective (guinea pig brain, [³H]DTG), NMDA glutamate (rat brain cortex), glutamate non- selective (rat brain, [³H]L-glutamate), glycine strychnine-sensitive (rat spinal cord, [³H] strychnine), Hi histamine central (guinea pig brain, [³H]pyrilamine), GABA_A agonist site (rat brain, [³H]muscimol), GABAA chloride channel (rat brain cortex, [³H]TBOB), estrogen (calf uterus, [³H]estradiol), progesterone (calf uterus, [³H]R- 5020), testosterone (rat ventral prostate, [ H]mibolerone), glucocorticoid (human Jurkat cells, [ H]dexamethasone), insulin (rat liver, [ IJinsulin), phorbol ester (mouse brain, [³H]PDBu); Voltage-activated ion channels: Ca²⁺ dihydropyridine- sensitive (rat brain cortex, [³H]nitrendipine); K⁺ (KATP) (syrian hamster pancreas, [³H]glyburide), Na⁺ site 2 (rat brain, [³H]batrachotoxin); Transporters: adenosine (guinea pig brain, [³H]NBTI binding), dopamine (human recombinant, [¹²⁵I]RTI-55 binding), serotonin (human recombinant, [¹²⁵I]RTI-55 binding), choline (rat brain, [ H]hemicholium binding), arachidonate (rat cortical neurons, [ Hjarachidonate uptake); ethanolamine (rat cortical neurons, [³H]ethanolamine uptake).

AM404 displaced the binding of the cannabinoid agonist [³H]WIN- 55212-2 from rat brain membranes with low affinity (EC₅₀ = 2 M), and did not activate CBl cannabinoid receptors when tested in vitro at 10 M (inhibition of forskolin-induced cyclic AMP accumulation in cortical neurons) or 30 M (stimulation of [³⁵S]GTP- -S binding in rat brain membranes. These results indicate that AM404 does no act as an agonist at CBl receptors. Moreover, AM404 did not prevent the inhibition of forskolin-induced cyclic AMP accumulation produced in cortical neurons by the application of WTN-55212-2, indicating that the drug does not act as a partial agonist on antagonist at CBl receptors. CB2 receptors do not appear to be expressed in the CNS (Ledent (1999) supra; Zimmer (1999) supra); thus, the interaction of AM404 with CB2 receptors was not investigated in the present experiments. Surgery. Implantation of stainless steel guide cannulae and icv injections were performed in lateral ventricles of male Wistar rats (>8 weeks old, 300-350 g) as described by Rodriguez de Fonseca (1996) J. Pharmacol. Exp. Ther. 276:56-64. AM404 (Tocris Cookson, Ballwin, MO; dissolved in 5 μl DMSO) or DMSO were inj ected via an 8-mm 30 gauge inj ector connected to a calibrated polyethylene- 10 tubing. Doses were not corrected for recovery after passage through the polyethylene tubing (see above), thus they represent an overestimate of the actual amount delivered to the tissue. Cannula placements were evaluated by injection of a blue dye, and only those rats with proper icv placements were included in the data analysis. Effects of AM404 on apomorphine-induced yawning. Apomorphine- induced yawning was measured in transparent plastic boxes (35x30x17 cm) following established procedures as described by, e.g., Yamada (1980) Psycopharmacology 67:39-43; Dourish (1989) Neuropharmacol. 28:1423-1425). AM404 (2 g per rat) or vehicle (DMSO, 5 1 per rat) were administered 5 min before subcutaneous (sc) injection of apomorphine (80 g per kg) or vehicle (aqueous 0.9% NaCI containing 40% DMSO, 0.2 ml per kg). Yawning was measured for a 30-min period following apomorphine injection. Intraperitoneal (ip) (IP) injections of AM404 (10 and 20 mg per kg), anandamide (0.1, 1 and 10 mg per kg) or vehicle (0.2 ml of aqueous 0.9% NaCI containing 10% DMSO) were done 30 min before apomorphine administration. DMSO alone had no effect on yawning.

Effects ofAM404 on basal and quinpirole-induced motor behaviors. Experiments were conducted as described by Giuffrida (1999) supra). The animals were housed in a room with controlled photoperiod (lights on: 8:00-20:00 h), and habituated to handling for a week before starting the experiments. All behavioural studies took place between 09:30-12:00 h. Locomotor activity was studied in an opaque open field (100x100x40 cm), the floor of which was marked with 20x 20 cm squares. The field was illuminated using a ceiling halogen light that was regulated to yield 350 lux at the center of the field. Rats were habituated to the field for 10 minutes the day before testing. On the experimental day, the animals were placed in the center of the open field and locomotor activity (number of lines crossed) was scored during five minutes. Behavior was tested 5, 30, 60 and 120 minutes after the injection of either vehicle or drugs. Spontaneous motor behavior was studied in a glass observation box (40x30x30 cm, one rat per box), and tested for 5 minutes at 5, 30, 60 and 120 minutes after drug injection. The tests were conducted in a sound- isolated room, illuminated with an indirect halogen light (125 lux). The behavior was videotaped on a video-cassette recorder. Animals were placed in the box 5 minutes before the onset of the testing period.

The following behavioural acts were scored: 1) immobility (defined as complete absence of observable movement), 2) number of rearing episodes, 3) time spent grooming; 4) sniffing activity, and 5) total oral activity (yawning, vacuous chewing and licking). Catalepsy by using the bar test was measured. At various times (0, 30, 60 or 120 min) after the injection of vehicle or drugs, the forepaws of test animals were positioned on a 10 cm-high bar, while keeping both hindpaws on the bench surface. The time the animals spent in an immobile position was measured. Tests were ended when the animals moved both forepaws onto the bench surface, or after 180 seconds of complete immobility. All behavioral measurements were scored by trained observers, blind to experimental conditions.

Effects ofAM404 in the hotplate test. The ability of AM404 to elicit analgesia was assessed by using the hot plate test. The rats were placed on a hot plate (55°C) and measured the latencies for the occurrence of nocifensive behaviors (paw licking or jumping). Test cutoff was 30 seconds. Effects ofAM404 on juvenile SHR rats. Juvenile male SHR (n=10) and

Wistar-Kyoto (n=15) rats (both from Charles River Italy) were used. The rats were kept two per cage in standard makrolon cages with water and food pellets (Mucedola, Italy) ad libitum. Four week-old rats were allowed a two-week acclimatization before testing. The experimental system was a Lat-maze, a 60x60x40 cm wooden box with a 30x30x40 cm plastic transparent smaller box inserted in the middle. Rats were allowed to explore the resulting corridor (60 cm long, 15 cm wide and 40 cm high). A set of four such boxes was located in a sound-attenuated room. The experimental box was illuminated by a white, cold 4 W lamp placed 60 cm above the floor in the center of the wooden cover, providing a 0.1 - 0.2 μW/cm². AM404 was dissolved in DMSO at a concentration of 1 mg per ml. Six week-old rats were exposed for 30 min to the Lat-maze after a single subcutaneous (sc) injection of AM404 (1 mg per kg) or vehicle (DMSO, lml/kg). Testing was carried out at the beginning of the light phase of the circadian cycle between 9:00 AM and 2:00 PM and the two members of the same cage were tested simultaneously to minimise the interference with the arousal state. Behavior was monitored by a CCD camera and stored on a tape recorder for off-line analysis by blind observers. The behavioral variables, i.e. the frequency of corner crossings as index of travel distance, duration of rearings on hindlimbs and leanings against the walls with one or both forepaws were visually monitored in 1- min blocks. The reliability index was quite high (r = 0.914; df= 198; p < 0.001). At the end of the test, the number of fecal boli was counted and the floor was carefully cleaned with a wet sponge. Frequency of corner crossings and frequency and duration of rearings were submitted to 3 -way factorial analysis of variance (ANOVA) rat line x treatment x time blocks (as dependent variable). Within-exposure changes in rearing duration were analysed by a 2-way ANOVA rat line x time blocks, as dependent variable. Planned comparisons between group means across days within-line or between line were made by the two-tailed t-test for paired or non-paired data, respectively. The effect of AM404 was assessed by separate 2-way ANOVA treatment x testing block (first vs. second phase of the test). The rejection level was set at P>0.05, two-sided. All animal procedures met the guidelines of the National Institutes of Health, detailed in the Guide for the Care of Laboratory Animals, and the European Community directives 86/609/EEC regulating animal research. Inhibition of [³H] anandamide transport. The ability of AM404 to inhibit [ H] anandamide transport has been documented in primary cultures of embryonic rat brain neurons and astrocytes (Beltramo 1997) Science 277:1094-1097) and in human astrocytoma cells (Piomelli (1999) supra). To determine whether [³H] anandamide transport is present in the adult CNS, [³H] anandamide accumulation in coronal slices of rat brain was investigated. Brain slices incubated in the presence of [³H]anandamide and SR141716A (a cannabinoid antagonist added to prevent binding of [³H] anandamide to CBl receptors) accumulated [³H] anandamide in a time- and temperature-dependent manner. As expected from a carrier-mediated process, the temperature-sensitive component of [³H]anandamide accumulation was prevented by nonradioactive anandamide, but not by other bioactive lipids (palmitylethanolamide, arachidonate and prostaglandin E₂) or by digoxin, a substrate of organic anion transport proteins. Replacement of extracellular Na⁺ with choline chloride or incubation with the metabolic inhibitor carbonyl cyanide 3-chlorophenyl hydrazone had no effect, suggesting a Na⁺- and energy-independent process. Moreover, [³H] anandamide uptake was prevented by AM404, but not by the anandamide amidohydrolase inhibitor, (E)-6-(bromomethylene)tetrahydro-3-(l-naphthalenyl)-2H- pyran-2-one (Beltramo (1997) FEBS Lett. 403:263-267). These findings indicate that [³H] anandamide accumulation in rat brain is mediated by a selective, Na⁺-independent transporter analogous to that found in rat cortical neurons and astrocytes in culture (Beltramo (1997) Science 277:1094-1097; Piomelli (1999) supra.

Inhibition of motor activity. Administration of AM404; 10 g per rat, intracerebroventricular (icv), but not of vehicle alone, 5 1 of dimethylsulphoxide (DMSO), caused a slow-onset reduction of motor activity that was statistically significant 60 min after drug injection. Cumulative immobility in the 120-min observation period was also significantly higher in AM404-treated animals than in controls. This response was prevented by the CBl antagonist SR141716A (1 mg per kilogram of body weight, ip injection 60 min before AM404), which did not affect movement when administered alone. The effect of AM404 was dose-dependent. No increase in immobility was observed after injection of a 0.4 g dose of AM404, whereas 2 g and 10 g doses were effective; times spent in immobility were, at 60 min: vehicle, 96.1+22.7 s (n=12); 0.4 g AM404, 77.7+26.2 s (n=8); 2 g AM404, 198.5+37.3 s (n=10); 10 g AM404, 175+18 seconds.

The hypokinetic actions of AM404 were reminiscent of those produced by administration of exogenous anandamide (Smith (1994) J. Pharmacol. Exp. Ther. 270:219-227). However, in sharp contrast with the latter, AM404 had no significant inhibitory effect on a variety of motor behaviors, including grooming, oral movements and sniffing. Although a trend toward decreased ambulatory activity was observed, this trend did not reach statistical significance under the conditions of the present experiments. Furthermore, AM404 did not elicit significant catalepsy or analgesia, two hallmarks of CBl receptor activation (Pertwee (1997) Pharmacol. Ther. 74:129-180). These data demonstrate that AM404 may act by interfering with anandamide clearance and by causing this endocannabinoid substance to accumulate slowly at a restricted number of release sites within the CNS. Distribution and selectivity ofAM404. The concentrations of AM404 reached in rat brain tissue after injection of a maximal dose of this compound (10 g, icv), were 1.4+0.5 M in striatum and 0.4_+0.3Min cortex (n=4, see Materials and Methods above). Comparable levels were measured in thalamus, hippocampus, brainstem and cerebellum. At such concentrations, AM404 strongly inhibits anandamide uptake by neurons and astrocytes, whereas it has no effect on 36 other drug targets: heterotrimeric GTP-binding protein-coupled receptors (including dopamine receptors), ligand-gated ion channels, amine uptake sites, and lipid transporters (see Materials and Methods). In particular, AM404 does not activate CBl receptors either in vitro or in vivo (see also Beltramo (1997) Science 277:1094- 1097; Calignano (1997) Eur. J. Pharmacol. 337:R1-R2; Calignano (1997) Eur. J. Pharmacol. 340:R7-R8), though it markedly enhances several effects of exogenously administered anandamide (Beltramo (1997) Science 277:1094-1097; Piomelli (1999) supra. Inhibition ofD₂-family receptor responses. In light of the observation that AM404 does not interact with D₂-family receptors, whether inhibition of anandamide transport may affect various behavioral responses produced by activation of these receptors was tested. AM404 in combination with either of two distinct dopamine receptor agonists: apomorphine and quinpirole, was administered. Low doses of the non-selective dopamine agonist apomorphine elicit a stereotypic yawning response that may be mediated by D -family receptors (Baraldi (1975) Riv. Farmacol. Terapia 6:771-772; Dourish (1989) Neuropharmacol. 28:1423-1425; Melis (1987) Brain Res. 415:98-104). The yawning induced by apomorphine (80 g per kg, sc) was strongly inhibited by AM404 (2 g per rat, icv), an effect that was prevented by the CBl antagonist SR141716A (0.2 mg per kg, intravenous (iv)). Similar inhibitions of the apomorphine response were observed after systemic injections of AM404 (10 and 20 mg per kg, ip). The inhibitory effect of systemic AM404 was antagonized by SR141716 and mimicked by anandamide (0.1-10 mg per kg, ip).

The selective D₂-family agonist quinpirole causes a biphasic motor response characterized by initial movement inhibition, which may be mediated by D - family autoreceptors, followed by a longer-lasting hyperactivity, possibly due to activation of postsynaptic D₂-family receptors (Eilam (1989) Eur. J. Pharmacol. 161:151-157). Administration of AM404 30 min before quinpirole (at a low dose of 0.25 mg per kg, sc) significantly enhanced the initial phase of locomotor inhibition elicited by quinpirole (at time = 5 min), whereas it reduced the subsequent phase of motor stimulation (at time = 120 min). A parallel effect of AM404 was observed on the time spent in immobility. This bimodal response is consistent with the finding that D -family receptors may stimulate anandamide outflow in vivo (Giuffrida (1999) supra), because neurally released anandamide is expected to act synergistically with D autoreceptors (which may mediate motor inhibition) but antagonistically with postsynaptic D receptors (which may cause motor activation) (Picetti (1997) Crit. Rev. Neurobiol. 11:121-142.

Reduction of genetic hyperactivity. Juvenile spontaneously hypertensive rats (SHR) are hyperactive, but not yet hypertensive, and show deficits of sustained attention in behavioral paradigms (Sagvolden (1993) Physiol. Behav. 54:1047-1055). These abnormalities have been associated with alterations in the activity of the mesocorticolimbic dopamine systems and with changes in dopamine receptor expression (Carey (1998) BBR 94: 173-185). To determine whether inhibition of anandamide transport affects hyperactivity in SHR, horizontal locomotor activity and duration of rearing episodes during exposure to a novel environment was measured after administration of AM404 or vehicle. In parallel tests, the effects of AM404 on age-matched Wistar-Kyoto (WKY) rats (the line from which SHR were selectively bred, see, e.g., Okamoto (1969) Pharmacol. Ther. 74:129-180) was examined. In control WKY rats, AM404 (1 mg per kg, sc, 30 min before testing) did not significantly affect rearing duration or horizontal locomotion in either the first (0- 15 min) or the second part (16-30 min) of the testing period. By contrast, the drug increased duration of rearing episodes and decreased horizontal activity in SHR during the second part of the test, in which vehicle-treated SHR fail to habituate to the novel environment and maintain inordinately high activity when compared to WKY controls (P < 0.05, ANOVA followed by Bonferroni's test). These results demonstrate that a low dose of AM404 can alleviate hyperactivity in SHR without significant effects on the normal motor behavior of progenitor WKY rats. In these studies, the anandamide transport inhibitor AM404 was used to investigate functional interactions between anandamide and dopamine in the confrol of motor activity. It was found that AM404 counteracts two characteristic responses mediated by activation of D₂-family receptors: apomorphine-induced 5 yawning and quinpirole-induced stimulation of motor behaviors. These effects are achieved at doses of AM404 that may elicit only a mild hypokinesia when the drug is administered alone, and may selectively inhibit anandamide transport in vitro. In addition, doses of AM404 identical to those used in the present study are able to produce a time-dependent increase in the levels of anandamide in peripheral blood. o Thus, these results are consistent with the hypothesis that anandamide released by stimulation of D -family receptors participates in the confrol of dopamine-induced psychomotor activation. In further support, it was found that AM404-sensitive anandamide transport is present in brain regions, such as cortex and striatum, that are crucially involved in the regulation of movement and that receive extensive 5 projections from midbrain dopamine-containing neurons (Albin (1989) TINS 12:366- 375).

CBl receptor agonists elicit abroad spectrum of behavioral responses that include catalepsy, analgesia, reduced movement and hypothermia (Pertwee (1997) supra). The finding that AM404 evokes only a moderate slow-onset 0 hypokinesia when it is administered alone demarcates the pharmacological profile of this anandamide transport inhibitor from those of direct-acting cannabimimetic drugs. This distinction may result from the ability of AM404 to enhance anandamide signaling in an activity-dependent manner by causing anandamide to accumulate in discrete regions of the CNS only when release of this endocannabinoid substance is 5 triggered by appropriate stimuli. In the absence of such stimuli, tonic anandamide release may be very low (Giuffrida(l 999) supra), accounting for the weak and slow- developing motor effects of AM404 in naϊve animals.

Accordingly, the pharmacological profile of AM404 as characterized by this invention provides an original strategy to correct behavioral abnormalities that 0 are generally associated with dysfunction in dopamine neurotransmission. As an initial test of this hypothesis, the effects of AM404 in SHR, a rat line in which hyperactivity and attention deficits have been linked to a defective regulation of mesocorticolimbic dopamine pathways, was examined. Administration of a low systemic dose of AM404 (1 mg per kg) normalizes motor activity in SHR with no overt motor effect in WKY controls (the strain from which SHR originate). The spectrum of pharmacological properties displayed by AM404 as provided by this invention and the ability of this drug to counteract potential manifestations of dopamine dysregulation demonstrate that anandamide transport is a valuable target for the novel neuropsychiatric medicines and methods of the invention.

Example 2: Systemic Administration of AM 404 Increases Plasma Levels of Anadamide The following example demonstrates that in vivo systemic administration of AM404 increases plasma levels of anadamide. These results support the finding that inhibitors of the inactivating transport of endogenous cannabinoids can ameliorate neuropsychiatric disorders. Specifically, these results support the finding that the behavioral effects of AM404 result from the ability of this compound to inhibit anandamide inactivation, thereby causing its accumulation in vivo. This study demonstrates the effects of systemic administration of an exemplary inhibitor of an inactivating transport of an endogenous cannabinoid substance, AM404, on the circulating levels of anandamide in rats. These results indicate that AM404 causes a time-dependent increase of peripheral anandamide, which is accompanied by a reduction in locomotor activity.

In these studies, fatty acyl chlorides (5,8,11,14- eicosatetraenoylchloride, hexadecanoylchloride and 9-cis-octadecenoylchloride) were from Nu-Check Prep (Elysian, MN); [²H₄]-labeled ethanolamine (isotopic atom enrichment = 98%) was from Cambridge Isotope Laboratories (Andover, MA); AM404 was from Tocris Cookson (Ballwin, MO); SR141716A was provided by RBI (Natick, MA) as part of the Chemical Synthesis Program of the NIMH (program no. N01MH30003). All solvents were from Burdick and Jackson (Muskegon, MI) and all other chemicals from Sigma (St. Louis, MO).

Synthesis ofunlabeled and f^'H^j '-labeled standards. Standard unlabeled and [²H₄]-labeled acylethanolamides (AEs) were synthesized by the reaction of the corresponding fatty acyl chlorides with unlabeled or [²H₄]-labeled ethanolamine respectively (Giuffrida and Piomelli, 1998; "Purification and high- resolution analysis of anandamide and other fatty acylethanolamides," in Lipid Second Messengers eds, pp 113-133, CRC Press LLC, Boca Raton, FL). This reaction results in the quantitative formation of AEs (Devane (1992) Science 258:1946-1949; Giuffrida and Piomelli (1998) supra), which were concentrated to dryness under a stream of N₂ and reconstituted in chloroform at a concentration of 20 mM. AE solutions were stored at -20 C until used. Identity and chemical purity (>98%) of the synthesized AEs were determined by TLC using chloroform/methanol/ammonia (85:15:1, vol/vol) as solvent system, and subsequently by HPLC/MS. Collection and preparation of rat plasma. Blood (2 ml) was collected from the heart of male Wistar rats anesthetized with methoxyflurane (Schering- Plough, Union, NJ) using a syringe filled with 1 ml of Krebs-Tris buffer (in mM: NaCI 136, KC1 5, MgCl₂ 1.2, CaCl₂2.5, glucose 10, Trizmabase 20; pH 7.4) containing 4.5 mM EDTA. Blood samples were drawn at 0, 30, 60, and 120 min after administration of a single dose of AM404 (10 mg-kg^~l intraperitoneal, i.p.), and centrifuged in Accuspin tubes (Sigma, St. Louis, MO) at 800xg, for 10 min at 22°C. After centrifugation, the plasma layers were recovered and spiked with 500 pmol of [²H₄]-labeled AEs. Plasma proteins were precipitated by adding cold acetone (-20°C, 1 vol) and removed by centrifugation at lOOOxg for 10 min. The supematants were flushed with a stream of N to evaporate acetone and subjected to lipid extraction with methanol/chloroform (1 :2, vol/vol). The recovered chloroform phases were evaporated to dryness under N₂, reconstituted in a mixture of chloroform/methanol (1:3, 80 1), and injected into the HPLC/MS for analysis and quantification. The estimated recoveries of anandamide, PEA and OEA were 98.7+0.2%, 78.1+0.8%, and 99.7+0.3%, respectively. In parallel experiments we also measured AM404 levels in plasma by HPLC/MS. However, because of the lack of an appropriate internal standard, the recovery of AM404 was consistently lower than that of the AEs (5.2+0.5%, n=4). HP 1100 Series HPLC/MS system™ equipped with a Hewlett- Packard octadecylsilica (ODS) Hypersil™ column (100x4.6 mm ID., 5 m) was used. Reversed-phase separations were carried out by using linear increases of methanol (B) in water (A) (25% A, 75% B for 2 min; 15% A, 85% B for 3 min; 5% A, 95% B for 20 min; 100% B for 5 min) at a flow rate of 0.5 ml/min as described in Giuffrida (2000) Anal. Biochem. 280:87-93. Under these conditions, analytes eluted from the column with the following retention times: anandamide, 15.4 min; PEA, 17.3 min; OEA, 18.4 min; AM 404, 15.9 min. MS analyses were performed in the positive ionization mode with an electrospray ion source. Capillary voltage was set at 3.0 kV, and fragmentor voltage was 80 V. Nitrogen was used as drying gas at a flow rate of 12 1/min. The drying gas temperature was set at 350 C and the nebulizer pressure at 50 psi. For quantitative analyses, diagnostic fragments corresponding to the protonated molecules ([M + H]⁺) and to the sodium adducts of the molecular ions ([M + Na]⁺) were followed in the selected ion monitoring (SIM) mode. System control and data evaluation were conducted using an on-line system software (HP Chemstation™).

Electrophoresis (PAGE)fRadiobinding assay. [³H] -anandamide (10 nM, 60 Ci/mmol) or [³H]-AM404 (10 nM, 200 Ci/mmol) (ARC, St. Louis, MO) were added to 10 mM potassium phosphate buffer (pH 7.4) containing either rat plasma (0.1 ml) or 70 M BSA (fraction V, Sigma, St. Louis, MO) and incubated for 30 min at 37 C. The incubations were stopped by adding 0.1 ml of a suspension of ice-cold Dextran VI (1 : 1 vol/vol, Sigma, St. Louis, MO). Dexfran was precipitated by centrifugation and 0.1 ml of supernatant were subjected to vertical slab gel electrophoresis (PAGE, 7.5% acrylamide) under non-denaturing conditions (Siegenthaler, 1990). The gel was cut into 2 mm bands, which were incubated for 3 h in 0.5 ml Solvable (Packard, Meriden, CT) at 50 °C. Radioactivity was measured by liquid scintillation counting.

Behavioral tests. The effects of AM404 (10 mg-kg"! intraperitoneal, i.p.), vehicle (0.9% saline containing 10% dimethyl sulfoxide, i.p.), and AM404 plus SR141716A (0.5 mg-kg^-1, i.p.), were studied on immobility and horizontal locomotor activity in two groups of male rats, differing in breeding, age and weight. The first group consisted of Wistar rats, 90-106 days/450-500 g (Charles River Laboratories, Wilmington, MA); the second group consisted of Sprague-Dawley rats, 56-70 days/250-300 g (Taconic, Germantown, NY). Motor activity was studied in a Digiscan photocell activity cage (42 x 42 x 30 cm) (Omnitech Electronics, Columbus, OH) equipped with 16 photocells (8 placed along the X, and 8 along the Y axis of the cage) and interfaced to a microcomputer (Digiscan Analyzer) that recorded activity automatically. On the experimental day, the animals were placed in the cage, and immobility (difference between sample time and time spent moving) and horizontal activity (distance traveled by the animal in a given sample period) were recorded at 5 min intervals. This procedure was performed at 5, 30, 60 and 120 min after the injection of either vehicle or drug. SR141716A was injected 60 min before the beginning of the experiment. All behavioral tests were conducted in a sound-isolated room, illuminated with an indirect halogen light (125 lux). For data analysis, results are expressed as means±s.e.m. Statistical significance was evaluated using ANOVA followed by the Dunnett's test.

Mass spectral properties ofAM404. As noted above, AM404 is a structural analog of anandamide characterized by a highly hydrophobic carbon chain and a polar carboxamido group carrying a hydroxyphenyl moiety. The mass spectral properties of AM404 were investigated by using reversed-phase LC/MS in a mobile phase of methanol/water. Mass spectra were acquired in the positive-ionization mode, because the total ion current (TIC) yielded by this ionization procedure was significantly higher than that obtained by negative ionization. The positive-ion electrospray spectrum of AM404 consisted of two main fragments: the protonated molecule ([M + H]⁺, m/z 396.3), and the Na⁺ adduct of the molecular ion ([M + Na]⁺, m/z 418.2). Both ions were accompanied by X+1 ¹³C isotope peaks of expected abundance (McLafferty and Turecek, (1993) Interpretation of Mass Spectra, University Science Books, Sausalito, CA). Additional informative fragments were m/z 287.2 ([M - 108]⁺, loss of NH-Ce^-OH) and m/z 245.2 ([M - 150]⁺, possibly corresponding to the loss of CH₂-CO-NH-C₆H -OH). Quantification of AM404 in plasma. AM404 analysis was carried out by monitoring the [M + Na]⁺ fragment (m/z 418.2) in the SEVI mode. This ion, although slightly less abundant than the [M + H]⁺ fragment, was selected because of its greater resolution from contaminating components present in plasma samples. For quantification purposes, a calibration curve was constructed by injecting into the LC/MS increasing amounts of AM404. The areas obtained from the integration of SIM peaks were plotted against the injected amounts. Under these conditions the MS responses were linear (r²= 0.93; n=3) over the range 0-250 pmol. Blood samples were collected by cardiac puncture from rats killed 30, 60, and 120 min following systemic (i.p.) injection of AM404 (a single dose of 10 mg/kg). After acetone precipitation of plasma proteins, lipids were extracted with chloroform/ methanol and analyzed by HPLC/MS. All plasma samples displayed a peak eluting from the column at the retention time of standard AM404 (15.9 min). Six independent experiments were conducted. The plasma concentrations of AM404, increased gradually reaching a maximal value (123+22 pmol/ml) at 60 min after injection, and declining thereafter. As expected of a hydrophobic molecule, the decline of AM404 in plasma was accompanied by a substantial accumulation of the compound in brain tissue. Quantification of anandamide in plasma. To determine whether the systemic administration of AM404 results in accumulation of endogenously produced anandamide, the plasma levels of anandamide after a bolus injection of AM404 (10 mg-kg^"1, i.p.) was measured. In the same samples, the levels of two additional fatty acid ethanolamides, PEA and OEA, which do not activate cannabinoid receptors, was measured. Quantification was carried out by monitoring the [M + Na]⁺ ions with the following m/z values: for anandamide and [²H₄]-anandamide, m/z 370.3 and 374.3, respectively; for PEA and [²H₄]-PEA, m/z 322.3 and 326.3; for OEA and [²H₄]-OEA, m/z 348.3 and 352.3. All plasma samples contained lipid components that eluted from the HPLC at the retention times expected for anandamide, PEA and OEA. In samples collected immediately (1 min) after AM404 administration, 2.8+0.3 pmol/ml of anandamide, 11+1.4 pmol/ml of PEA and 10.3+2.1 pmol/ml of OEA (n=12) was measured. These levels are identical to those observed in rats that received no drug injection. Anandamide levels in plasma increased gradually after AM404 administration, and were significantly different from controls 60 min (5.5+1.4 pmol/ml, n=7) and 120 min (8.1+1.8 pmol/ml, n=7) after injection of the drug. By contrast, the plasma levels of PEA were not affected by AM404, while those of OEA showed a trend towards accumulation that was statistically different from controls only 120 min after injection (19.6+3.1 pmol/ml).

Binding of anandamide andAM404 to plasma proteins. Anandamide and AM404 were detectable in plasma only when proteins were first denaturated with cold acetone, suggesting that these lipids may be tightly associated to plasma proteins. To test this hypothesis, [³H]-labeled anandamide was incubated in plasma or in a solution containing BSA (70 M), and analysed by PAGE under non-denaturing conditions (2-mm sections of the gel were cut, and radioactivity was measured by liquid scintillation counting). All plasma samples showed a significant radioactive peak that comigrated on the gel with albumin. Similar results were obtained after electrophoresis of either plasma or BSA solutions incubated with [³H]-AM404. Effects ofAM404 on motor behavior. Systemic administration of AM404 (10 mg/kg, i.p.; in Wistar rats) caused a time-dependent decrease in motor activity, which was measured either as increase of time spent in total immobility or as reduction of horizontal locomotion. This effect was statistically significant 30 and 60 min after drug administration. Comparable inhibitory effects on motor behaviors were observed in a second group of animals differing from the previous in breeding, age and weight (Sprague-Dawley rats). In the latter group we also tested the effects of the selective CBl receptor antagonist SR141716A. Administration of SR141716A (0.5 mg/kg, i.p.) 60 min before AM404 did not cause any change in basal motor activity, but reversed the effects of AM404 on immobility and locomotor activity.

In summary, these data demonstrate that systemic administration of AM404 increases the concentration of circulating anandamide in a time-dependent fashion. These changes were accompanied by an increase of AM404 levels in plasma, which remained as high as 70.5 pmol/ml for at least two hours after drug administration. As expected of hydrophobic molecules, it was found that both anandamide and AM404 bind to a protein in plasma, which was identified as albumin by non-denaturing PAGE. This sequestration may contribute both to the kinetics of accumulation of anandamide and AM404 in plasma, and to their distribution in other tissues. To further investigate the biochemical effects of AM404 in vivo, the plasma levels of PEA and OEA was monitored (PEA and OEA are two fatty acylethanolamides that are produced through the same biosynthetic mechanism of anandamide, but do not serve as substrates for the anandamide transporter). AM404 administration did not significantly affect the levels of PEA, but caused a slow increase of circulating OEA statistically significant 120 min after AM404 injection. Since OEA is not transported by anandamide carrier, a possible interpretation of this result is that AM404 may inhibit an as-yet uncharacterized transporter of OEA. Alternatively, OEA elevation may result from the interference of AM404 with anandamide amidohydrolase (AAH), of which OEA represents a substrate. This possibility is supported by the fact AM404 inhibits AAH activity in brain membranes with an IC₅₀ = 5.3+0.9 M (mean±s.e.m., n=3). However, administration of the potent AAH blocker, AM374 (see, e.g., Gifford (1999) Eur. J. Pharmacol. 383: 9-14) had no effect on circulating anandamide levels, although it significantly increased the levels of OEA 30 min after drug application. Taken together, these results suggest that blockade of AAH activity is unlikely to participate in the elevation of anandamide in plasma, but may cause the accumulation of other fatty acid acylethanolamides.

In parallel with its ability to increase anandamide levels in plasma, AM404 also induced a time-dependent inhibition of motor activity. This hypokinesia, which is reminiscent of that observed after anandamide administration (see, e.g., Fride (1993) Eur. J. Pharmacol. 231: 313-314; Smith (1994) J. Pharmacol. Exp. Ther. 270: 219-227; Romero (1995) Brain Res. 694: 223-232), was reversed by the CBl receptor antagonist SR141716A. The reversal of AM404 actions cannot be accounted for by the inverse agonist properties of SR141716A (see Landsman (1997) Eur. J. Pharmacol. 334: R1-R2), as this compound had no effects on motor activity when given alone at 0.5 mg/kg. Since the motor inhibition produced by AM404 was achieved at a dose that also caused accumulation of anandamide in peripheral blood, and given the inability of AM404 to activate cannabinoid receptors, these results are consistent with the hypothesis that AM404 produced its behavioral effects by protecting endogenous anandamide from transport-mediated inactivation. Although the concentrations reached by anandamide in plasma (approximately 10 nM) are insufficient to activate cannabinoid receptors (Kd = 50 nM and 1.6 M for CBl and CB2 receptor, respectively) (see, e.g., Pertwee (1997) Pharmacol. Ther. 74:129-180), AM404 may cause anandamide to accumulate in brain tissue to an extent that is sufficient to cause biological effects. This would explain why the motor inhibition elicited by AM404 takes place before significant accumulation of anandamide in plasma is observed. However, the sources of plasma anandamide following AM404 administration are still unknown. Indeed, anandamide production has been demonstrated not only within the central nervous system, but also in peripheral cells, such as macrophages and platelets (Schmid (1997) Methods Enzymol. 189: 299-307; Wagner (1997) Nature 390: 518-521).

Example 3: The Functional Role of High Affinity Anadamide Transport as Revealed by Inhibition of that Transport The following example demonstrates a high-affinity transport system present in neurons and astrocytes that has a role in anandamide inactivation by removing this lipid mediator from the extracellular space and delivering it to intracellular metabolizing enzymes.

Anandamide (arachidonylethanolamide), an endogenous ligand for central cannabinoid receptors, is released from neurons on depolarization and rapidly inactivated. Anandamide inactivation is not completely understood, but it may occur by transport into cells or by enzymatic hydrolysis. The compound N-(4- hydroxyphenyl) arachidonylamide (AM404) was shown to inhibit high-affinity anandamide accumulation in rat neurons and astrocytes, an indication that this accumulation resulted from carrier-mediated transport. Although AM404 did not activate cannabinoid receptors or inhibit anandamide hydrolysis, it enhanced receptor- mediated anandamide responses in vitro and in vivo. The data indicate that carrier- mediated transport is important for termination of the biological effects of anandamide and represents a novel drug target. Anandamide is an endogenous lipid that activates brain cannabinoid receptors and mimics the pharmacological effects of 9-tetrahydrocannabinol, the active principle of hashish and marijuana . In humans, such effects include euphoria, calmness, oneiroid states and drowsiness. Depolarized neurons release anandamide through a mechanism that may involve the calcium-dependent cleavage of a phospholipid precursor in neuronal membranes. Like other modulatory substances, extracellular anandamide is thought to be rapidly inactivated, but the exact nature of this inactivating process is still unclear. One possible pathway is hydrolysis to arachidonic acid and ethanolamine, catalyzed by a membrane-bound fatty acid amide hydrolase (FAAH) highly expressed in rat brain and liver. Nevertheless, the low FAAH activity found in brain plasma membranes indicates that this enzyme may be intracellular, a possibility that is further supported by sequence analysis of rat FAAH (see, e.g. Cravatt (1996) Nature 384:83. Although anandamide could gain access to FAAH by passive diffusion, transfer rate is expected to be low, because of the molecular size of this lipid mediator. In that other lipids including polyunsaturated fatty acids and prostaglandin E2 (PGE2) enter cells by carrier-mediated transport, it is possible that anandamide uses a similar mechanism. Indeed, the existence of a rapid, saturable process of anandamide accumulation into neural cells has been reported (Di Marzo (1994) supra). This accumulation may result from the activity of a transmembrane carrier, which may thus participate in termination of the biological actions of anandamide. Accordingly, the instant invention developed drug inhibitors of anandamide transport and demonstrated their pharmacological properties in cultures of rat cortical neurons and astrocytes.

The accumulation of exogenous [^H] anandamide by neurons or astrocytes fulfills several criteria of a carrier-mediated transport. It is a rapid process that reaches 50% of its maximum within approximately 4 min. Furthermore, [^H] anandamide accumulation is temperature-dependent and saturable. Kinetic analyses were performed using cultures of cortical neurons were prepared from rat embryos (Stella (1995) J. Neurosci. 15:3307) or astrocytes (Beltramo (1997) FEBS Lett. 403:263) and were used after 4 to 6 days and 21 to 25 days in vitro, respectively.

Accumulation of [^H] anandamide (221 Ci/mmol, New England Nuclear, Wilmington, DE) was measured by incubating the cells (six-well plates) for various times in Krebs buffer (NaCI, 136 mM; KCl, 5 mM; MgCl2, 1.2 mM; CaCl2, 2.5 mM; glucose, 10 mM; Trizmabase, 20 mM; pH 7.4 at 37°C) containing [^H] anandamide (0.45 nM, brought to 100 nM with non radioactive anandamide). Incubations were stopped by aspirating the media, cells were rinsed with Krebs buffer containing bovine serum albumin (BSA, 0.1% w/v), and subjected to extraction with methanol and chloroform. Radioactivity in the extracts was measured directly or after fractionation of cell lipids by thin-layer chromatography (Beltramo (1997) FEBS Lett.

403:263). For kinetic analyses, the neurons were incubated for 4 min at 37°C in the presence of 10 to 500 nM anandamide containing 0.05 to 2.5 nM [^H] anandamide.

We subtracted non-specific accumulation (measured at 0° to 4°C) before determining kinetic constants by Lineweaver-Burk analysis. Kinetic analyses revealed that accumulation in neurons can be represented by two components of differing affinities (lower affinity: Km 1.2 M, Nmax 90.9 pmol/min per mg of protein; higher affinity: K_m 0.032 M, Nmax 5.9 pmol/min per mg of protein). The higher affinity component may reflect a binding site, however, as it is displaced by the cannabinoid receptor antagonist, SR-141716-A

(100 nM). In astrocytes, ?H] anandamide accumulation is represented by a single high-affinity component (K_m 0.32 M, Nmax 171 pmol min per mg of protein). Such apparent Km values are similar to those of known neurotransmitter uptake systems (see, e.g., Barker and Blakely, In Psychopharmacology: The Fourth Generation of Progress, Bloom and Kupfer, Eds., Raven Press, New York, 1995, pp. 321-334) and are suggestive therefore of high-affinity carrier-mediated transport. To characterize further this putative anandamide transport cortical astrocytes in culture were used. As expected from a selective process, the temperature-sensitive component of [^H] anandamide accumulation was prevented by non radioactive anandamide, but not by palmitoylethanolamide, arachidonate, prostanoids or leukotrienes. Replacement of extracellular Na⁺ with N- dimethylglucosamine or choline had no effect (as percentage of control: N- dimethylglucosamine, 124+12%; choline, 98+14%; mean+SEM, n = 6) suggesting that [^H] anandamide accumulation is mediated by a Na⁺-independent mechanism, which has been observed with other lipids (Bito (1996) Acta Physiol. Scand.

156:501). Moreover, inhibition of FAAH activity by treating the cells with (E)-6- (bromomethylene)tetrahydro-3-(l-naphthalenyl)-2H-pyran-2-one (25 M) or Hnoleyl trifluoro methylketone (15 M) (Koutek (1994) J. Biol. Chem. 269:22937) had no effect. This indicates that anandamide hydrolysis did not provide the driving force for anandamide transport into astrocytes within the time frame of our experiments. Finally, the cannabinoid receptor agonist WTN-55212-2 (1 M) and antagonist SR- 141716- A (10 M) also had no effect, suggesting that receptor internalization was not involved.

A primary criterion for defining carrier-mediated transport is pharmacological inhibition. To identify inhibitors of anandamide transport, we first examined compounds that are known to prevent the cellular uptake of other lipids, such as fatty acids (phloretin, 50 M), phospholipids (verapamil 100 M, quinidine 50 M), or PGE2 (bromcresol green, 0.1 to 100 M) (Bito (1994) Cell 77:1071). Among the compounds tested only bromcresol green interfered with anandamide transport, albeit with limited potency and partial efficacy. Bromcresol green inhibited [3H] anandamide accumulation with IC50 (concentration needed to produce half- maximal inhibition) of 4 M in neurons and 12 M in astrocytes, and acted non competitively. In astrocytes, Nmax values for [^H] anandamide accumulation were 200 pmol/min per mg of protein without bromcresol green, and 111 pmol/min per mg of protein with bromcresol green (10 M). Apparent Km values were 0.24 M and 0.25 M, respectively (n = 6). Moreover, bromcresol green had no significant effect on the binding of [ H]WTΝ-55212-2 to rat cerebellar membranes (Ki = 22 M),

FAAH activity in rat brain microsomes (IC50 > 50 M), and uptake of [-1H] arachidonate or [-1H] ethanolamine in astrocytes (121+13% and 103+12% respectively at 50 M bromcresol green, n = 3). Displacement of [^H] WIN-55212-2 binding (40 to 60 Ci/mmol; New England Nuclear) to rat cerebellar membranes (0.1 mg/ml) was determined as described by Kuster (1993) J. Pharmacol. Exper. Therap. 264:1352. Non specific binding was measured in the presence of 1 M non radioactive WTN-55212-2. FAAH activity was measured in rat brain particulate fractions, as described by Beltramo (1997) supra. The uptake of [^H] arachidonate (Amersham, 200 Ci/mmol; 5 nM brought to 100 nM) and [^H] ethanolamine

(Amersham, 50 Ci/mmol; 20 nM brought to 100 nM) was determined on cortical astrocytes for 4 min as described above. The control uptake for [³H] arachidonate was 16729+81 dpm/well and for [^H] ethanolamine it was 644+100 dpm/well (n = 6). The sensitivity to bromcresol green, which blocks PGE2 transport, raised the question of whether anandamide accumulation occurred by means of a PGE2 carrier. That this is not the case was shown by the lack of [^H] PGE2 accumulation in neurons or astrocytes (neurons or astrocytes were incubated for 4 min at 37°C in Krebs buffer containing [³H] PGE2 (0.67 nM brought to 100 nM with nonradioactive PGE2; 171 Ci/mmol, New England Nuclear). After rinsing with Krebs buffer/BS A, the cells were lipid extracted; radioactivity was counted in the extracts. On average, neurons contained 245+65 dpm/well and astrocytes 302+20 dpm/well; non specific accumulation in astrocytes at 0 to 4 °C was 355+28 dpm well (n = 6)). It was also demonstrated by the inability of PGE2 to interfere with [³H] anandamide accumulation. Previous results indicating that expression of PGE2 transporter mRNA in brain tissue is not detectable further support this conclusion, see Kanai (1995) Science 268:866.

To search for more potent anandamide fransport inhibitors, a series of structural analogs of anandamide were synthesized and tested (Khanolkar (1996) J. Med. Chem. 39:4515). From this screening, the compound N-(4- hydroxyphenyl)arachidonylamide (AM404) was selected. AM404 was both efficacious and relatively potent; IC50 was 1 M in neurons and 5 M in astrocytes. AM404 acted as a competitive inhibitor, suggesting that it may serve as a transport substrate or pseudosubstrate. By contrast, at the concentrations tested AM404 had no effect on FAAH activity (IC50 > 30 M) or on uptake of [³H] arachidonate or [³H] ethanolamine (102+4% and 96+14% respectively at 20 M AM404, n = 6). Furthermore, a positional isomer of AM404, N-(3-hydroxyphenyl) arachidonylamide (AM403), was significantly less effective than AM404 in inhibiting transport. These data provide pharmacological evidence for the existence of a specific anandamide transporter and suggest, (i) that neurons and astrocytes may act synergistically in the brain to dispose of extracellular anandamide and, (ii) that the transport systems in these two cell types may differ kinetically and pharmacologically.

The identification of these inhibitors allowed examination of whether transmembrane transport participates in terminating anandamide responses mediated by cannabinoid receptor activation. Cannabinoid receptors of the CBl subtype are expressed in neurons, where they are negatively coupled to adenylyl cyclase activity. It was found that in cultures of rat cortical neurons the cannabinoid receptor agonist WTN-55212-2 inhibited forskolin-stimulated cyclic AMP accumulation (pmol per mg of protein; control: 39+4; 3 M forskolin: 568_+4; forskolin plus 1 M WTN-55212-2: 220+24) and this inhibition is prevented by the antagonist, SR-141716-A (1 M)

(555+39 pmol per mg of protein, n = 9). Anandamide produced a similar effect, but with a potency that was approximately 20 times lower (IC 1 M) than that expected from its binding constant for CBl cannabinoid receptors (Ki 50 nM)(l). The transport inhibitor AM404 binds to CBl receptors with low affinity (Ki 1.8 M) and did not reduce cyclic AMP levels when applied at 10 M. Nevertheless, the drug markedly enhanced the effects of anandamide, increasing the potency (10 times greater) and decreasing the threshold (100 times lower), an effect that was prevented by SR-141716-A. Thus, a concentration of anandamide that was below threshold when applied alone (0.3 M) produced an almost maximal effect when applied together with AM404. Bromcresol green and AM403, which were less effective than AM404 in inhibiting anandamide transport, were also less effective in enhancing the anandamide response. Furthermore, the decreases in cyclic AMP levels produced by WTN-55212-2 (which stimulates CBl receptors, but is not subject to physiological clearance) or glutamate (which stimulates metabotropic receptors negatively coupled to adenylyl cyclase and is cleared by a selective transporter) are not affected by any of the anandamide transport inhibitors tested.

These results demonstrate that pharmacological blockade of carrier- mediated transport protects anandamide from physiological inactivation, enhancing the potency of anandamide to nearly that expected from its affinity for CBl cannabinoid receptors in vitro. To find out whether this potentiation occurs in vivo, we tested the effects of AM404 on the antinociceptive activity of anandamide in mice. Intravenous anandamide (20 mg/kg) elicited a modest but significant analgesia, as measured by the hot plate test (Fride (1993) Eur. J. Pharmacol. 231:313; Smith (1994) Pharmacol. Exp. Therap. 270:219) (P < 0.05, Student's t test); this analgesia disappeared 60 min after injection and was prevented by SR-141716-A The hot plate test (55.5 °C) was carried out on male Swiss mice (25-30 g, Nossan, Italy) following standard procedures, e.g., as described by Porreca (1994) J. Pharmacol. Exp. Therap. 230:341. Anandamide and AM404 were dissolved in 0.9% NaCI solution containing 20% dimethyl sulfoxide, and injected intravenously at 20 mg/kg and 10 mg/kg, respectively. To determine whether cannabinoid receptors participate in the effect of anandamide, anandamide (20 mg/kg i.v.) or anandamide plus SR141716-A (2 mg/kg, subcutaneously) was administered to two groups of 6 mice each. In mice that received anandamide alone, latency to jump increased from 21.7+1.5 s to 30.7+0.8 s (P < 0.05, ANON A) 20 min after injection. By contrast, in mice that received anandamide plus SR141716-A, the latency to jump was not affected (19.6+3.1 s). Administration of AM404 (10 mg kg, intravenously) had no antinociceptive effect within 60 min of injection, but significantly enhanced and prolonged anandamide- induced analgesia (P < 0.01, Student's t test).

In conclusion, these findings indicate that a high-affinity transport system present in neurons and astrocytes has a role in anandamide inactivation by removing this lipid mediator from the extracellular space and delivering it to intracellular metabolizing enzymes such as FAAH. Therefore, the invention has identified selective inhibitors of anandamide transport and novel therapeutic agents.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for ameliorating a neuropsychiatric disorder in a patient in need thereof by inhibiting the inactivating transport of an endogenous cannabinoid substance, wherein the method comprises administration to the patient of a pharmaceutical composition able to inhibit the inactivating transport of an endogenous cannabinoid, wherein the administration of the pharmaceutical composition is in an amount sufficient to inhibit the inactivating transport of an endogenous cannabinoid substance and to ameliorate the neuropsychiatric disorder in the patient.

2. The method of claim 1 , wherein the endogenous cannabinoid substance comprises anandamide.

3. The method of claim 1 , wherein the endogenous cannabinoid substance comprises 2-arachidonylglycerol.

4. The method of claim 1 , wherein the pharmaceutical composition comprises a compound consisting essentially of (i) a hydrophobic carbon chain moiety comprising at least one nonconjugated cis double bond in the middle of the chain, linked to (ii) a polar carboxamido or carboxyester moiety, linked to (iii) a nonionizable polar head group.

5. The method of claim 4, wherein the hydrophobic carbon chain moiety has one to six nonconjugated cis double bonds.

6. The method of claim 4, wherein the hydrophobic carbon chain moiety has a length of C-l 8 to C-22.

7. The method of claim 4, wherein the compound is compound 1 , compound 2, compound 3, compound 4, compound 5, compound 6, compound 13, compound 20 or compound 21 or an equivalent thereof or a mixture thereof.

8. The method of claim 4, wherein the pharmaceutical composition comprises N-(4-hydroxyphenyl)arachidonamide (AM404), N-(3- hydroxyphenyl)arachidonamide or an equivalent thereof or a mixture thereof.

9. The method of claim 4, wherein the nonionizable polar head group further comprises an alkyl group in the form of an S isomer.

10. The method of claim 9, wherein the pharmaceutical composition comprises an S-l '-methyl anandamide (compound 21).

11. The method of claim 1 , wherein the pharmaceutical composition comprises a compound consisting essentially of (i) a hydrophobic carbon chain moiety comprising at least one nonconjugated cis double bond in the middle of the chain, linked to (ii) a polar carboxamido or carboxyester moiety, linked to (iii) a head group as set forth in compound 11, compound 12, compound 18, compound 19, compound 20, compound 21, compound 22, compound 23, compound 28, compound 29, compound 30, compound 31, compound 32, compound 33 or compound 34 or an equivalent thereof or a mixture thereof.

12. The method of claim 11, wherein the hydrophobic carbon chain moiety has one to six nonconjugated cis double bonds.

13. The method of claim 11 , wherein the hydrophobic carbon chain moiety has a length of C-l 8 to C-22.

14. The method of claim 11 , wherein the head group is a polar nonionizable head group with a hydrogen-donating hydroxyl group.

15. The method of claim 11 , wherein the head group is a polar nonionizable head group with a hydrogen-accepting group.

16. The method of claim 15, wherein the hydrogen-accepting group is an ether containing group or a phenolic group.

17. The method of claim 1 , wherein the pharmaceutical composition comprises oleylethanolamide or oleamide or an equivalent thereof or a mixture thereof.

18. The method of claim 1 , wherein inhibiting the inactivating transport comprises inhibiting the inactivating transport of an endogenous cannabinoid substance.

19. The method of claim 18, wherein inhibiting the inactivating transport of an endogenous cannabinoid substance causes accumulation of the endogenous cannabinoid substance in a synaptic site.

20. The method of claim 1 , wherein inhibiting the inactivating transport of an endogenous cannabinoid substance counteracts the effects of dopamine hyperactivity.

21. The method of claim 1 , wherein neuropsychiatric disorder is at least in part caused or mediated by dopamine hyperactivity.

22. The method of claim 21 , wherein the dopamine hyperactivity is in a central nervous system (CNS) region.

23. The method of claim 1 , wherein neuropsychiatric disorder is schizophrenia.

24. The method of claim 1 , wherein neuropsychiatric disorder is a schizoaffective disorder.

25. The method of claim 1, wherein neuropsychiatric disorder is a schizophreniform disorder.

26. The method of claim 1 , wherein neuropsychiatric disorder is borderline personality disorder.

27. The method of claim 1, wherein neuropsychiatric disorder is attention-deficit hyperactivity disorder.

28. The method of claim 1 , wherein neuropsychiatric disorder is an autism spectrum disorder.

29. The method of claim 1 , wherein neuropsychiatric disorder is Tourette's syndrome.

30. The method of claim 1, wherein neuropsychiatric disorder is a psychoactive substance-induced organic mental disorder or a psychoactive substance use disorder.

31. The method of claim 1 , wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient comprising an aqueous solution or a lipid based solution.

32. The method of claim 1 , wherein the pharmaceutical composition is administered by an oral, a parenteral, a sublingual, a fransmucosal or a transdermal route.

33. A pharmaceutical composition comprising a compound able to inhibit the inactivating transport of an endogenous cannabinoid substance and a pharmaceutically acceptable excipient.

34. The pharmaceutical composition of claim 33, wherein the compound consists essentially of (i) a hydrophobic carbon chain moiety comprising at least one nonconjugated cis double bond in the middle of the chain, linked to (ii) a polar carboxamido or carboxyester moiety, linked to (iii) a nonionizable polar head group.

35. The pharmaceutical composition of claim 34, wherein the hydrophobic carbon chain moiety has one to six nonconjugated cis double bonds.

36. The pharmaceutical composition of claim 34, wherein the hydrophobic carbon chain moiety has a length of C-l 8 to C-22.

37. The pharmaceutical composition of claim 34, wherein the compound is compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 13, compound 20 or compound 21 or an equivalent thereof or a mixture thereof.

38. The pharmaceutical composition of claim 34, wherein the compound comprises N-(4-hydroxyphenyl)arachidonamide (AM404), N-(3- hydroxyphenyl)arachidonamide or an equivalent thereof or a mixture thereof.

39. The pharmaceutical composition of claim 34, wherein the hydroxyl ethyl head group further comprises an alkyl group in the form of an S isomer.

40. The pharmaceutical composition of claim 39, wherein the pharmaceutical composition comprises an S-l'-methyl anandamide (compound 21).

41. The pharmaceutical composition of claim 33, wherein the compound consists essentially of (i) a hydrophobic carbon chain moiety comprising at least one nonconjugated cis double bond in the middle of the chain, linked to (ii) a polar carboxamido or carboxyester moiety, linked to (iii) a head group as set forth in compound 11, compound 12, compound 18, compound 19, compound 20, compound 21, compound 22, compound 23, compound 28, compound 29, compound 30, compound 31, compound 32, compound 33 or compound 34 or an equivalent thereof or a mixture thereof.

42. The pharmaceutical composition of claim 41, wherein the hydrophobic carbon chain moiety has one to six nonconjugated cis double bonds.

43. The pharmaceutical composition of claim 41 , wherein the hydrophobic carbon chain moiety has a length of C- 18 to C-22.

44. The pharmaceutical composition of claim 41 , wherein the head group is a polar nonionizable head group with a hydrogen-donating hydroxyl group.

45. The pharmaceutical composition of claim 41 , wherein the head group is a polar nonionizable head group with a hydrogen-accepting group.

46. The pharmaceutical composition of claim 45, wherein the hydrogen-accepting group is an ether containing group or a phenolic group.

47. The pharmaceutical composition of claim 33, wherein the compound comprises oleylethanolamide or oleamide or an equivalent thereof or a mixture thereof.

48. The pharmaceutical composition of claim 33, wherein the concentration of the compound in the pharmaceutically acceptable excipient is between about 0.1 mg per kg and about 10 mg per kg of body weight

49. The pharmaceutical composition of claim 33 , wherein the pharmaceutically acceptable excipient is an aqueous solution or a lipid based solution.

50. The pharmaceutical composition of claim 33 formulated for administration by an oral, a parenteral, a sublingual, a transmucosal or a transdermal route.

51. A kit comprising a pharmaceutical composition and printed material, wherein the pharmaceutical composition comprises a compound and a pharmaceutically acceptable excipient, wherein the compound is able to inhibit the inactivating transport of an endogenous cannabinoid substance, and wherein the printed matter comprises instructions for use of the pharmaceutical composition to ameliorate a neuropsychiatric disorder in a patient in need thereof.