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Aromatic L-amino acid decarboxylase deficiency is a genetic disorder of enzyme loss with decreased neurotransmitter synthesis, and it is characterized by symptoms of impaired motor development and cognitive function, hypotonia, dystonia, and oculogyric crises. Though symptomatic severity varies, the majority of patients experience severe motor impairments, including an inability to sit, stand, or walk. One approved therapy for Aromatic L-amino acid decarboxylase deficiency involves intraputaminal delivery of an adeno-associated virus packaging the human Aromatic L-amino acid decarboxylase enzyme (hAADC) cDNA. The objective of this study in monkeys was to determine the acceptability of ICV/IT as minimally invasive dosing options by evaluating hAADC biodistribution and expression following intraputaminal, intracerebroventricular (ICV), or intrathecal (IT, lumbar) administration. Results show that all routes produced comparable CSF transgene levels and were well-tolerated. The intraputaminal route yielded the highest levels of transgene-derived mRNA expression in the putamen, caudate, and globus pallidus, while expression levels in the spinal cord and dorsal root ganglia (DRG, a target of special toxicological concern) were undetectable. In contrast, the highest transgene levels in ICV/IT groups were observed in the spinal cord and DRG, but levels were too low to result in expression in the putamen, caudate, and globus pallidus. Unlike ICV/IT, the intraputaminal route produced no transgene in blood, suggesting a lower likelihood of off-target toxicities. Additionally, intraputaminal dosing resulted in the lowest anti-AAV2 antibody (anti-drug antibody) levels. Together, these data demonstrate the superiority of intraputaminal administration over ICV/IT routes in achieving AAV2-hAADC transgene DNA distribution and mRNA expression in target therapeutic areas while minimizing risk of toxicity.

Nonclinical development strategies for gene therapies are unique from other modalities. The European Federation of Pharmaceutical Industries and Associates (EFPIA) Gene Therapy Working Group surveyed EFPIA member and nonmember pharmaceutical and biotechnology companies about their current practices for designing and implementing nonclinical toxicology studies to support the development of viral vector-delivered in vivo gene therapies. Compiled responses from 17 companies indicated that these studies had some variability in species selection, study-design elements, biodistribution, immunogenicity or genomic insertion assessments, safety pharmacology, and regulatory interactions. Although there was some consistency in general practice, there were examples of extreme case-by-case differences. The responses and variability are discussed herein. Key development challenges were also identified. Results from this survey emphasize the importance for harmonization of regulatory guidelines for the development of gene-therapy products, while still allowing for case-by-case flexibility in nonclinical toxicology studies. However, the appropriate timing for a harmonized guidance, particularly with a platform that continues to rapidly evolve, remains in question., Graphical Abstract, The European Federation of Pharmaceutical Industries and Associates Gene Therapy Working Group conducted a survey on current practices associated with nonclinical toxicity studies conducted to support the development of in vivo viral vector-delivered gene therapies. Responses provide insight on studies conducted, endpoints evaluated, and challenges faced by biopharmaceutical/biotechnology companies.

This commentary provides an overview of the putamen as an established target site for gene therapy in treating aromatic l‐amino acid decarboxylase (AADC) deficiency and Parkinson’s disease, two debilitating neurological disorders that involve motor dysfunction caused by dopamine deficiencies. The neuroanatomy and the function of the putamen in motor control provide good rationales for targeting this brain structure. Additionally, the efficacy and safety of intraputaminal gene therapy demonstrate that restoration of dopamine synthesis in the putamen by using low doses of adeno‐associated viral vector serotype 2 to deliver the hAADC gene is well tolerated. This restoration leads to sustained improvements in motor and nonmotor symptoms of AADC deficiency and improved uptake and conversion of exogenous l‐DOPA into dopamine in Parkinson’s patients., This Commentary provides an overview of putamen‐targeted gene therapies for treating aromatic l‐amino acid decarboxylase (AADC) deficiency and Parkinson’s disease.

Loperamide is a μ-opioid receptor agonist commonly used to treat diarrhea and often available as an over-the-counter medication. Recently, numerous reports of QRS widening accompanied by dramatic QT interval prolongation, torsades de pointe arrhythmia, and death have been reported in opioid abusers consuming large amounts of the drug to produce euphoria or prevent opiate withdrawal. The present study was undertaken to determine the mechanisms of this cardiotoxicity. Using whole-cell patch clamp electrophysiology, we tested loperamide on the cloned human cardiac sodium channel (Nav1.5) and the two main repolarizing cardiac K(+) channels cloned from the human heart: KvLQT1/minK and the human ether-a-go-go-related gene (hERG) channel. Loperamide inhibited Nav1.5 with IC50 values of 297 and 239 nM at holding potentials of -90 and -70 mV, respectively. Loperamide was weakly active on KvLQT1/minK producing 17 and 65 % inhibition at concentrations of 1 and 10 μM, respectively. Conversely, loperamide was found to be a very high affinity inhibitor of the hERG channel with an IC50 value of 89 nM at room temperature and 33 nM when measured at physiological temperature. The QRS and QT interval prolongation and the attending arrhythmias, produced by loperamide, derive from high affinity inhibition of Nav1.5 and especially hERG. Since the drug has been widely available and safely used as directed for many years, we believe that the potent inhibition loperamide possesses for cardiac ion channels has only been uncovered because of the excessive misuse of the drug as a consequence of the recent opioid abuse epidemic.

BACKGROUND The covalent modification of interferon (IFN) alpha2b with monomethyoxy polyethylene glycol (PEG) reduces its clearance rate and increases its half-life. High doses of interferon (IFN) alpha2b have previously been shown to affect maintenance of pregnancy in rhesus monkeys. Given the role of ovarian hormones in reproductive function and pregnancy, this study was conducted to assess the effects of PEG-IFNalpha2b or IFNalpha2b (comparative control) on ovarian hormones and menstrual cyclicity in cynomolgus monkeys. In addition, the potential for reversibility of PEG-IFNalpha2b or IFNalpha2b-related observations was assessed. METHODS Monkeys were administered 3,105 microg/m(2) human recombinant (hr) IFNalpha2b or 52, 262, or 4,239 microg/m(2) PEG-hr-IFNalpha2b every other day for one menstrual cycle, followed by a post-dose period of up to two menstrual cycles. RESULTS Monkeys administered 3,105 microg/m(2) hr-IFNalpha2b or 52, 262, or 4,239 microg/m(2) PEG-hr-IFNalpha2b exhibited transient decreases in food consumption, leukocyte and erythrocyte parameters. Monkeys administered 3,105 microg/m(2) hr-IFNalpha2b exhibited lengthened menstrual cycles that were associated with a delay in reaching peak ovarian hormone levels and lower respective peak concentrations. Similarly, monkeys administered 4,239 microg/m(2) PEG-hr-IFNalpha2b exhibited lengthened menstrual cycles and a delay in reaching peak ovarian hormone levels and slightly lower respective peak concentrations. Post-dosing menstrual cycle length, estradiol and progesterone profiles exhibited evidence of recovery in both the hr-IFNalpha2b and the high-dose PEG-hr-IFNalpha2b groups. CONCLUSIONS Administration of hr-IFNalpha2b or PEG-hr-IFNalpha2b at high doses to cynomolgus monkeys resulted in similar effects on menstrual cycles, estradiol and progesterone profiles, and exhibited evidence of reversibility upon cessation of dosing. These results suggest that the previously observed high-dose IFNalpha-related effects on the maintenance of pregnancy in monkeys are likely the result of altered ovarian function.

Mu opiate agonists morphine, fentanyl and meperidine are administered short-term to pediatric patients, from the neonatal period through adolescence. However, there has been no assessment of the effect of age on the analgesic efficacy or the concentration–response relationship for these opioids in human pediatric patients. Few studies in animals have correlated opioid anti-nociception and tissue levels of these opioids commonly administered to pediatric patients. The present study was conducted to examine the role of age on opioid anti-nociceptive potency and efficacy and brain and plasma opioid levels to provide predictive information on the effect of opioids in developing humans. Administration of trace amounts of tritiated drug with anti-nociceptive doses of unlabeled drug was used for the assessment of anti-nociception in the tail-flick test and for the measurement of brain and plasma drug equivalent levels in postnatal rats (PND 3–21). Morphine and fentanyl were completely efficacious in all postnatal ages examined, although age-related differences in drug potency, as well as, differences in brain and plasma levels were observed. There was a good correlation between morphine ( r =0.96) and fentanyl ( r =0.89) ED 50 values and their respective brain and plasma EC 50 equivalent levels. Meperidine had limited efficacy in young rats (PND 3–9) but was completely efficacious in older rats (PND 14–17). However, PND 21 rats experienced tonic–clonic seizures which limited its efficacy to 70% anti-nociception. Our data suggest that pharmacokinetics, the development of the blood–brain barrier and ontogeny of opioid receptor function may play important roles in the sensitivity of postnatal rats to mu receptor agonists.

(Dimethylheptyl)anandamide [(16,16-dimethyldocosa-cis-5,8,11,14-tetraenoyl)ethanolamine ] (17a) and its amide analogs were synthesized by Wittig coupling of a ylide derived from a fragment of arachidonic acid. These amides were compared to the endogenous cannabinoid receptor ligand arachidonylethanolamide (anandamide, 2a) and its amide analogs in pharmacological assays for potential enhancement of cannabimimetic activities. The receptor affinity to rat brain membranes of the dimethylheptyl (DMH) analogs increased by an order of magnitude in most comparisons to the corresponding anandamides in displacement assays versus the cannabinoid agonist [3H]CP 55,940 or antagonist [3H]SR141716A, for which rank order differences in affinity were observed. An order of magnitude enhancement of potency with comparable or higher efficacy in behavioral assays in the mouse tetrad of tests of cannabinoid activity was observed in 17a versus 2a. In contrast, no enhancement in potency for the pentyl to DMH side chain exchange was seen in the mouse vas deferens assay. The data indicate a structural equivalence between classical plant cannabinoids and 2a as well as different receptor-ligand interactions that characterize multiple receptor sites or binding modes.

The synthesis of both side chain epimers of 1′-( 4 ), 2′-( 5 ), 3′-methyl-( 6 ) and 4′-methyl-Δ 8 -tetrahydrocannabinol ( 7 ) has been carried out. The synthetic approach entailed the acid catalyzed condensation of the appropriate substituted resorcinol with menthadienol to provide the Δ 8 -THC analogue. Both isomers of 1′-( 4 ) and 2t-Δ 8 -THC ( 5 ) were more potent than Δ 8 -THC, both in vitro and in vivo . The 3′-methyl isomers ( 6 ) were approximately equal in potency to Δ 8 -THC, and 4t-methyl-Δ 8 -THC was less potent. There was relatively little difference in potency between the epimers of 4 , 5 , and 6 .

This investigation was directed at characterizing some of the pharmacological properties of 6'-cyanohex-2'-yne-delta 8-tetrahydrocannabinol (O-823), a compound with high affinity for cannabinoid binding sites (Ki = 0.77 nM). In mouse vasa deferentia, O-823 behaved as a potent partial cannabinoid CB1 receptor agonist (EC50 = 0.015 nM). In the guinea-pig myenteric plexus preparation, it antagonized WIN 55.212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholino)methyl]pyrrolo-[1,2,3-de ]-1, 4-benzoxazin-6-yl](1-naphthyl)methanone] and CP 55.940 [(-)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl ) cyclohexan-1-ol] with Kd values of 0.65 and 0.27 nM, respectively. After in vivo delta 9-tetrahydrocannabinol pretreatment. the sensitivity of vasa deferentia to O-823-induced inhibition of electrically evoked contractions was reduced by 127-fold. 3.162 nM O-823 was inhibitory in unpretreated vasa deferentia but antagonized CP 55,940 in pretreated tissues (Kd = 0.26 nM). O-823 is probably an antagonist in the myenteric plexus preparation and delta 9-tetrahydro-cannabinol pretreated vasa deferentia but a partial agonist in unpretreated vasa deferentia because the first two of these preparations contain fewer receptors than the third.

A series of 3-alkyl-2'-yne (side chain) acetylenic analogs of delta 9-THC were synthesized and evaluated for in vitro and in vivo activity. Analogs were evaluated for receptor affinity in a [3H]CP-55,940 displacement assay and for in vivo pharmacological activity in a mouse procedure utilizing a tetrad of measures. These compounds represent a preliminary exploration of the consequences of restricting the flexibility of the side chain regarding cannabimimetic activity. All analogs proved to have receptor affinities (4-11 nM) that were five to ten times greater than that observed for delta 9-THC. However, the in vivo activities of these compounds varied greatly. All analogs proved to possess the greatest potency for production of antinociception, with activity similar to or less than that observed for the production of hypomotility, hypothermia, and catalepsy. The most potent analog 11b exhibited an ED50 of 0.031 mg/kg in the tail-flick procedure, with values in other measures being between 0.5 and 1.0 mg/kg. The least active compound (11c), though still possessing a KI of 11 nM, exhibited ED50 values of 3.1 and 9.3 mg/kg for tail-flick and temperature procedures, as well as 41 and 48 mg/kg for ring-immobility and spontaneous locomotor activity, respectively. This profile (high receptor affinity but low in vivo potency) would normally be suggestive of a compound with antagonist properties (at least for immobility and activity measures). It is unclear why these acetylenic analogs were so potent in vitro, while only one (11b) exhibited the degree of in vivo potency anticipated based upon comparison to values for delta 9-THC. It is possible these side chain modifications do not interfere with receptor recognition, but limit receptor activation or second messenger signal transduction. Regardless, it is clear these novel analogs provide a basis for the further exploration of the cannabinoid receptor pharmacophore.

The basic premise underlying the cannabinoid pharmacophore is that at least three functional groups are involved in the interaction between the ligand and the receptor and that these functional groups in delta 9-THC comprise (a) C11, (b) the phenolic hydroxyl, and (c) the side chain. In order to assess the relative importance of the C11 position and the side chain, a series of C11 substituted analogs were prepared which contained a dimethylheptyl side chain. Consistent with previous studies, incorporation of a dimethylheptyl side chain dramatically enhanced both pharmacological potency in mice and receptor affinity. Incorporation of a hydroxy at C11 along with this branched side chain resulted in an extremely potent cannabinoid with ED50S of 0.01, 0.04, 0.16 and 0.04 mumol/kg in depression of spontaneous activity, reduction in body temperature, antinociception, and immobility, respectively. This compound was also very potent as a discriminative stimulus in a drug discrimination procedure and exhibited an extended duration of action. Its high affinity for the cannabinoid receptor (Ki = 400 pM) was consistent with this pharmacological potency. Incorporation of an oxo rather than a hydroxy reduced potency somewhat, although this analog was much more potent than delta 9-THC in most behavioral assays. The most striking observation was that incorporation of a carboxylic acid to form 11-nor-delta 9-THC-DMH-9-carboxylic acid did not eliminate pharmacological activity. This analog was as potent as delta 9-THC. The improbability that all three of the functional groups are interacting in a similar fashion with the receptor provides further support that the C11 position is not an essential requirement for activity. On the other hand, it is possible that substituents in the C9 region are interacting somewhere within or near the same site, but differently.

The design and synthesis of a series of 1-alkyl-3-(1-naphthoyl)pyrroles is described. Molecular modeling studies were employed to aid in the design of these compounds. During the course of the synthesis the Friedel-Crafts reactions of N-aryl sulfonyl pyrroles were reinvestigated. The title compounds (4) were subjected to pharmacological evaluation and the data obtained have enabled these pyrroles to be classified as cannabinoids.

Both C9 epimers of Δ7-THC (3 and 4) have been synthesized from Δ8-THC methyl ether (12). Hydroboration of 12 gave a mixture of stereoisomeric 8-hydroxyhexahydrocannabinols. The 8α-ol was converted into the 9β-methyl epimer (4) of Δ7-THC, while the 8β-ol was converted into 3. Molecular modeling indicated that 4 should have significant cannabinoid activity, while 3 was predicted to be weakly active. These predictions were borne out by both in vitro and in vivo pharmacology.

Molecular modeling has been employed to design a new group of cannabimimetic 1-alkyl-2-methyl-3-(1-naphthoyl)indoles. Cannabinoid activity was evaluated in vivo in the mouse and in vitro by determining the binding to the cannabinoid receptor. Maximum activity was found for the 1-butyl, pentyl and hexyl analogs. A rationalization for the alignment of these indoles with traditional cannabinoids is presented.

The design and synthesis of a series of 1-alkyl-3-(1-naphthoyl)pyrroles is described. Molecular modeling studies were employed to aid in the design of these compounds. During the course of the synthesis the Friedel-Crafts reactions of N-aryl sulfonyl pyrroles were reinvestigated. The title compounds (4) were subjected to pharmacological evaluation and the data obtained have enabled these pyrroles to be classified as cannabinoids.

The synthesis of a variety of novel 10-substituted cannabidiol (CBD) and 11- or 12-substituted delta 8-tetrahydrocannabinol (delta 8-THC) analogues containing amino, alkylamino, azido, or a N,N-bis(2-chloroethyl)amino functional group is described, as well as their pharmacological evaluation in mice. These analogues, which possess only a portion of the full pharmacological spectrum of activity of delta 9-THC, indicate that cannabinoid-mediated reduction of spontaneous locomotor activity, hypothermia, antinociception, and/or catalepsy need not be produced simultaneously, possibly suggesting the existence of more than one mechanism of action. The 10-substituted CBD analogues 3, 4, and 5 with an ethylamino, propylamino, or azido functional group, respectively, proved to be largely inactive, except for the production of central nervous system (CNS) depression concomitant with toxicity. Toxicity and CNS depression may be related phenomena in these nitrogenous compounds since 12-amino and 12-ethylamino analogues (8 and 11) of delta 8-THC also proved to be very toxic. Antinociceptive and hypothermic responses (without reduction of motor activity) were observed at a dose of 10 mg/kg of the 11-ethylamino analogue (9) of delta 8-THC, while a dose of 50 mg/kg of the nitrogen mustard 11-[N,N-bis(2-chloroethyl)amino]-delta 8-THC (12) was necessary to produce any observable pharmacological effect. When selected analogues were evaluated for antagonistic properties, they failed to attenuate the effects of delta 9-THC. Some nitrogen mustard analogues were capable of producing minimal pharmacological effects after either peripheral or direct CNS administration; however, these analogues also failed to attenuate the effects of delta 9-THC either immediately after administration or 24-48 h later.

The two water-soluble cannabinoids 1-[(4-morpholino) butyryloxy]-delta 8-tetrahydro-cannabinol (MB-delta 8-THC) and 5'-trimethylammonium (TMA)-delta 8-THC, as well as structurally similar compounds, were evaluated for cannabimimetic activity in the mouse (locomotor activity, tail-flick antinociception, rectal temperature, and ring-immobility) and dog (static-ataxia) procedures. MB-delta 8-THC possesses full cannabimimetic activity and is approximately equipotent to delta 8-THC. 5'-TMA-delta 8-THC only possesses partial cannabimimetic activity in that it is inactive in the ring-immobility and static-ataxia procedures. However, this analog is potent in other respects. All alterations at the 5' position do not necessarily produce this spectrum of effects, as evidenced by the pharmacological activity of 5'-bromo-delta 8-THC, 5'-OH-delta 8-THC acetate, and 5'-N-dimethyl-delta 8-THC.

The separation of the mood-altering effects of cannabinoids from their therapeutic effects has been long sought. Results reported here for a series of C-9 analogs of the cyclic ether O,2-propano-delta 8-tetrahydrocannabinol (O,2-propano-delta 8-THC) point to the C-1 position in classical cannabinoids as a position for which CB2 subtype selectivity occurs within the cannabinoid receptors. O,2-Propano-11-delta 8-THC, O,2-propano delta 9,11-THC, O,2-propano-9-oxo-11-nor-hexahydrocannabinol (O,2-propano-9-oxo-11-nor-HHC), and O,2-propano-9 alpha- and O,2-propano-9 beta-OH-11-nor-HHC were synthesized and evaluated in radioligand displacement assays for affinity at the CB1 and CB2 receptors and in the mouse vas deferens in vitro assay and the mouse tetrad in vivo assay for cannabinoid activity. Evaluation of binding affinity at the CB1 and CB2 receptors revealed that each compound possesses a modest increased affinity for the CB2 receptor. Analogs which contained an oxygen attached to C-9 (i.e., oxo and hydroxy derivatives) showed the highest affinity and selectivity for CB2 (for O,2-propano-9-oxo-11-nor-HHC, Ki(CB1) = 90 nM, Ki(CB2) = 23 nM, selectivity ratio 3.9; for O,2-propano-9 beta-OH-11-nor-HHC, Ki(CB1) = 26 nM, Ki(CB2) = 5.8 nM, selectivity ratio 4.5). Each compound was found to produce a dose-dependent inhibition of electrically-evoked contractions of the mouse isolated vas deferens when administered at submicromolar concentrations. This inhibition could readily be prevented by the selective CB1 cannabinoid receptor antagonist SR-141716A. The analogs exhibited unique in vivo profiles with O,2-propano-delta 9,11-THC exhibiting antinociception with reduced activity in three other in vivo measures and O,2-propano-9 beta-OH-HHC exhibiting lack of dose responsiveness in all measures. The CB2 selectivities in the O,2-propano analogs may be due to differences in solvation/desolvation that occur when the ligands enter the CB1 vs CB2 binding site. Alternatively, the CB2 selectivities may be a results of an amino acid change from a hydrogen bond-accepting residue in CB1 to a hydrogen bond-donating residue in CB2.

Several novel 4-alkyloxy-aminoalkyl indole derivatives 3 were synthesized from 4-benzyloxyindole (1). Alkylation of 1 with 4-(2-chloroethyl)morpholine (NaH/HMPA) formed 2. Deprotection using palladium hydroxide on carbon/hydrogen followed by alkylation with the appropriate alkyl bromide gave the target compounds 3b-3j. In the synthesis of 3i and 3j, the appropriate alkyl bromides 13 and 17 were prepared from the commercially available 1-naphthylethyl bromide 9 using the chain lengthening sequences as shown in Scheme 3. In receptor binding assay and in vivo testing, the long chain alkoxy compounds 3g and 3h (Ki = 127 nM) showed affinity for the CB1 receptor which was approximately 16-35-fold less than that of WIN 55,225. However, the pharmacological profile of 3h mimics that of WIN 55,212. An examination of the SAR of these analogues shows that translocating the napthyl group in AAIs from the C-3 position to C-4 via an oxygen (ether linkage) decreases activity which is in contrast to previous findings that a naphthylcarbonyl at C-4 retains activity. The present work points to the importance of the role of a keto group in the interaction with the receptor. Molecular modeling work suggests that, although reasonable superposition of key structural features between delta 9-THC and AAIs can be made, the overlay is not straightforward. The present study also illustrates the difficulty in accommodating AAIs into the cannabinoid pharmacophore and it seems likely that a unique pharmacophore will need to be developed. Only then will the similarities to and differences from the classical cannabinoid pharmacophore be clearly delineated.

alpha-Benzyl-N-methylphenethylamine (BNMPA), an impurity of illicit methamphetamine synthesis, has previously been reported to produce convulsions in mice without affecting spontaneous locomotor activity or altering methamphetamine-induced increases in spontaneous activity. In this study the in vitro effects of BNMPA on a variety of neuronal receptor types was determined to better characterize the pharmacological actions of this novel compound. BNMPA and N-demethyl-BNMPA fully displaced the dopamine transporter selective ligand [3H]CFT (2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane) from rat striatal membranes with Ki values (mean +/- S.E.M) of 6.05 microM +/- 0.15 and 8.73 microM +/- 1.66, respectively. BNMPA also inhibited [3H]dopamine uptake into striatal synaptosomes with an IC50 value of 5.1 +/- 1.4 microM. The basal efflux of [3H]dopamine from striatal slices was slightly enhanced by BNMPA only at concentrations > or = 100 microM. BNMPA had no effect on [3H]norepinephrine efflux from hippocampal slices. BNMPA displaced tritiated paroxetine and prazosin binding from rat cortical membranes with Ki values of 14.5 microM and 11.7 microM respectively. In electrophysiological studies, BNMPA (100 microM) had no significant effects on either GABAA Cl- currents in cultured neurons or non-NMDA glutamate receptors expressed in oocytes. However, BNMPA significantly inhibited NMDA-stimulated currents in oocytes expressing the NR1/2A or NR1/2C receptor subunit combinations (IC50 values = 24.6 +/- 1.8 and 24.0 +/- 1.5 microM, respectively). This inhibition was rapid, reversible and voltage-dependent. These results indicate that BNMPA has multiple sites of action in the CNS that could be important in modulating a variety of behavioral effects upon exposure to this synthetic byproduct of illicit methamphetamine synthesis.

delta 8-Tetrahydrocannabinol (delta 8-THC) is a naturally occurring cannabinoid with a characteristic pharmacological profile of in vivo effects. Previous studies have shown that modification of the structure of delta 8-THC by inclusion of a nitrogen-containing functional group alters this profile and may alkylate the cannabinoid receptor, similar to the manner in which beta-funaltrexamine (beta-FNA) alkylates the micro-opioid receptor. Two novel analogs of delta 8-THC were synthesized: a nitrogen mustard analog with a dimethylheptyl side chain (NM-delta 8-THC) and a cyano analog with a dimethylpentyl side chain (CY-delta 8-THC). Both analogs showed high affinity for brain cannabinoid receptors and when administered acutely, produced characteristic delta 9-THC-like effects in mice, including locomotor suppression, hypothermia, antinociception and catalepsy. CY-delta 8-THC shared discriminative stimulus effects with CP 55,940; for NM-delta 8-THC, these effects also occurred, but were delayed. Although both compounds attenuated the effects of delta 9-THC in the mouse behavioral tests, evaluation of potential antagonist effects of these compounds was complicated by the fact that two injections of delta 9-THC produced similar results, suggesting that acute tolerance or desensitization might account for the observations. NM-delta 8-THC, but not CY-delta 8-THC, attenuated the discriminative stimulus effects of CP 55,940 in rats several days following injection. Hence, addition of a nitrogen-containing functional group to a traditional cannabinoid structure does not eliminate agonist effects and may produce delayed attenuation of cannabinoid-induced pharmacological effects.

In this study, we report the isolation from canine intestines of 2-arachidonyl glycerol (2-Ara-Gl). Its structure was determined by mass spectrometry and by direct comparison with a synthetic sample. 2-Ara-Gl bound to membranes from cells transiently transfected with expression plasmids carrying DNA of either CB 1 or CB 2 —the two cannabinoid receptors identified thus far—with K i values of 472 ± 55 and 1400 ± 172 nM, respectively. In the presence of forskolin, 2-Ara-Gl inhibited adenylate cyclase in isolated mouse spleen cells, at the potency level of Δ 9 -tetrahydrocannabinol ( Δ 9 -THC). Upon intravenous administration to mice, 2-Ara-Gl caused the typical tetrad of effects produced by THC: antinociception, immobility, reduction of spontaneous activity, and lowering of the rectal temperature. 2-Ara-Gl also shares the ability of Δ 9 -THC to inhibit electrically evoked contractions of mouse isolated vasa deferentia; however, it was less potent than Δ 9 -THC.

Anandamide (arachidonylethanolamide), isolated from porcine brain, has been shown to bind to the cannabinoid receptor and also to produce cannabimimetic activity in pharmacological assays. This study examined structure-activity relationships in alkylated anandamide analogs. The analogs were evaluated for their ability to displace [3H]CP-55,940 in a filtration binding assay using rat brain membranes in the presence and absence of the enzyme inhibitor phenylmethylsulfonyl fluoride (PMSF). Behavioral activity was assessed by the ability of the analogs to produce hypomotility and antinociception. Methylations at carbons 2 and 1 produced compounds stable in the absence of PMSF with similar affinities and behavioral activity as anandamide. Addition of larger alkyl groups at these positions or nitrogen methylation reduced receptor affinity and behavioral potency. These results indicate that methylations at specific carbons of anandamide confer stability in vitro.

11-Hydroxy-3-(1',1'-dimethylheptyl)hexahydrocannabinol (1) was synthesized from the known cannabimimetic analog (+/-)-nabilone. Racemic 1 was resolved by HPLC on a semipreparative CHIRALCEL OD column (Daicel, Inc.), and pharmacological activities of the individual enantiomers were evaluated in the mouse model. The (-)-enantiomer was found to be much more potent than the (+)-enantiomer in all the four measures with the potency ratios in the production of catalepsy (RI), hypoactivity (SA), hypothermia (RT), and antinociception (TF) being 93, 143, 186, and 322, respectively. The racemic 11 alpha-OH diastereomer (2), a reaction side product, was also evaluated in the mouse model. Only small differences in the pharmacological activity of racemic 1 and 2 were found in the above four measures.

Despite a large body of research directed at assessing the effects of perinatal cannabinoid exposure, little is known about the development of the cannabinoid receptor. Recent advances, including the cloning of the cannabinoid receptor, have afforded us the opportunity to plot the postnatal ontogeny of the cannabinoid receptor and its mRNA in whole brain using the methods of receptor binding and RNA blot hybridization, respectively. Our results indicate that cannabinoid receptor mRNA is present at adult levels as early as postnatal day 3. The Bmax, on the other hand, increases almost fifty percent with increasing postnatal age, while the affinity does not change. The Hill coefficients for all ages studied were approximately 1. These findings suggest the possibility of a developmental progression for cannabinoid receptor development with receptor mRNA appearing first, followed by a period of rapid proliferation of the receptors themselves.

The primary goal of this research was to synthesize a series of ether analogues of the cannabinoid drug class and to evaluate their agonist and antagonist pharmacological properties in either the mouse or the rat. Agonist and antagonist activity was evaluated in mice using a multiple-evaluation procedure (locomotor activity, tail-flick latency, hypothermia, ring immobility) and activity in rats determined in a discriminative stimulus paradigm. Additionally, novel analogues were evaluated for their ability to bind to the THC receptor site labeled by 3H-CP-55,940. None of the cannabinoid analogues were capable of attenuating the effects of delta 9-THC (3 mg/kg) in either the rat (doses up to 10 mg/kg) or in the mouse (doses up to 30 mg/kg). It also appears that the compounds with minimal in vivo activity are not mixed agonist/antagonists. These data would suggest that the phenolic hydroxyl is important for receptor recognition (binding) and in vivo potency. Additionally, cannabinoid methyl ethers previously considered inactive have been found to produce limited activity. Lastly, data suggest that delta 9,11-THC is more potent than previous reports indicated, and does possess pharmacological activity.

The compound 9-beta-hydroxy-hexahydrocannabinol [(−)-9β-OH-HHC] was designed to fit a combined theoretical profile of an analgesic cannabinoid (equatorial alcohol at C-9, phenol at C-1 and a C-3 side chain) with reduced psychoactivity (axial C-9 substituent which protrudes into the α face). (−)-9β-OH-HHC was synthesized by the addition of methyl Grignard to 9-oxo-11-nor-HHC. Its α epimer was obtained by the regiospecific epoxide ring opening of 9α, 10α-epoxy-HHC acetate. (−)-9β-OH-HHC and (−)-9α-OH-HHC were each evaluated in a battery of tests in mice and were found to be 10–25 times less potent than (−)-trans-Δ9-tetrahydrocannabinol (Δ9-THC) in all tests including the tail flick test for antinociception (analgesia). Molecular mechanics calculations [MMP2(85)] revealed that, in the global minimum energy conformation of (−)-9β-OH-HHC, the axial methyl at C-9 protrudes into the α face of the molecule, while the axial hydroxyl at C-9 in (−)-9α-OH-HHC protrudes into this same face. These calculations also identified a higher energy carbocyclic ring (twist) conformer of each in which there is no protrusion of a C-9 substituent of the carbocyclic ring into the α face. The minimal activity of both compounds is attributed to these higher energy forms. It is concluded that protrusion of a C-9 substituent into the α-face of the molecule is associated with reduced cannabinoid analgesia, as well as with reduced cannabinoid psychopharmacological activity.

The chronic abuse of many drugs produces both marked tolerance and physical dependence. Marked tolerance to cannabis has been observed in experimental animals and humans. However, reports of physical dependence, as characterized by significant withdrawal symptomatology upon cessation of chronic cannabis exposure, has not been well established or clearly defined in any species. The abuse potential of cannabis is more readily observed in humans than in experimental animal investigations. This may be due to the physiochemical characteristics of cannabis or delta 9-THC, which complicates this type of animal experimentation. It is more likely that the greatest dangers of cannabis abuse involve the pharmacological effects of the drug upon the central nervous system and other organs, rather than the development of dependence.

A series of novel mercapto and thioacetyl derivatives of Δ 8 -tetrahydrocannabinol ( Δ 8 -THC, 5 ), and cannabidiol ( 1 ) were synthesized. Treatment of 10-hydroxy-cannabidiol ( 2 ) with thioacetic acid in the presence of a complex of diisopropylazodicarboxylate and triphenylphosphine in dry THF gave 10-thioacetyl-cannabidiol ( 3 ). Further treatment with LiAlH 4 converted 3 into 10-mercapto-cannabidiol ( 4 ). Using a similar sequence, 11-mercapto- Δ 8 -THC ( 8 ) was synthesized from the metabolite 11-hydroxy- Δ 8 -THC ( 6 ) via the corresponding thioacetyl derivative ( 7 ). Similarly, the 12β-thioacetyl derivative ( 10 ) of Δ 8 -THC was prepared. Although 8 is a derivative of a pharmacologically active cannabinoid ( 6 ), 8 proved to be inactive in 4 different pharmacological evaluations in the mouse. Similarly, 10 and 4 were also inactive. Additionally, 7 was inactive except for the production of hypothermia, but was more than 3-fold less potent than Δ 8 -THC. None of these cannabinoids was able to antagonize the effects of Δ 9 -THC. These data indicate that there are specific structural requirements for the production of cannabimimetic activity, which tends to suggest that activity is determined, in part, via specific molecular interactions such as those observed in receptor-, enzyme-, ion channel-, or other protein-mediated events.

In vivo and in vitro binding studies, both in whole brain and in selected areas, indicate that non-identical (+)- and (-)-NANM sites exist in the mouse brain, and each exhibits a different regional distribution. The in vivo binding of (+)-/sup 3/H-NANM was found to be saturable at pharmacologically relevant doses, and represents a relatively small (10 - 22%) portion of total brain (+)-/sup 3/H-NANM concentrations. The in vivo binding of (+)-/sup 3/H-NANM was selectively displaced by (+)-NANM and PCP, and more sensitive to haloperidol and (+)-ketocyclazocine than the (-)-/sup 3/H-NANM site. The in vivo binding of (-)-/sup 3/H-NANM was selectively displaced by (-)-NANM, and more sensitive to naloxone and (-) ketocyclazocine than the (+)-/sup 3/H-NANM site, and insensitive to PCP. This study indicates that the investigation of NANM binding sites is possible using in vivo binding techniques, and that each isomer apparently binds, in the mouse brain, to a single class of distinct sites. 32 references, 4 figures, 2 tables.

Studies were conducted to determine whether single or combination treatments of charcoal, paraffin, cholestyramine, and/or ammonium chloride (NH4Cl), would alter the rotarod-measured motor dysfunction induced by 10 to 90 mg/kg of phencyclidene (PCP). Additionally, the effect of NH4Cl/charcoal treatment of the biodisposition of 50 mg/kg PCP was evaluated in order to assess whether amelioration of behavioral effects could be correlated to alterations in brain levels, plasma levels, and/or the renal clearance of PCP and metabolites. NH4Cl/charcoal treatment proved more effective at reducing intoxication than either treatment singly, though effectiveness was reduced by larger doses of PCP. NH4Cl/charcoal treatment reduced intoxification by 40, 16, and 21% at PCP doses of 10, 25, and 50 mg/kg. However, the reduction in motor dysfunction observed at 25 and 50 mg/kg PCP was greater than the sum of the individual treatments. In contrasts, the effect of combined NH4Cl and charcoal treatment on the biodisposition of 50 mg/kg PCP is not synergistic, but appears instead to be due simply to the additive effects of the individual treatments. Thus the amelioration of PCP intoxication cannot be fully explained by alterations in PCP biodisposition.

The effects of the enantiomers of 11-hydroxy- Δ 8 -tetrahydrocannabinol-dimethylheptyl (11-OH- Δ 8 -THC-DMH) on spontaneous activity, rectal temperature, tail-flick latency, and catalepsy were studied in mice and in the dog static-ataxia model to determine the relative potency of each enantiomer. The (−)-enantiomer was active in all tests between 3–100 μg/kg, while the (+)-enantiomer was inactive at 30 mg/kg in the mouse and 1 mg/kg in the dog. The (−)-enantiomer was 100–800 times more potent than Δ 9 -THC in the mouse. The high degree of enantioselectivity and potency are suggestive of an interaction at a specific site such as a receptor.

Regulation of the rate-limiting step in dopamine (DA) synthesis was estimated in striatal synaptosomes by measuring the rate of hydroxylation of L-4-[3H]phenylalanine, a substrate of tyrosine hydroxylase (TH). DA inhibited hydroxylation with an IC50 of 0.2 microM. The concentration-response curve of DA-induced inhibition was not affected by the presence of 1 microM chlorpromazine, a phenothiazine DA antagonist. Sulpiride and haloperidol, DA antagonists of the benzamide and butyrophenone classes respectively, also failed to alter the inhibition of substrate hydroxylation by 1 microM DA, even at concentrations up to 10 microM. In contrast, a parallel 15 fold shift to the right in the concentration-response curve of DA-induced inhibition of hydroxylation was obtained when 10 microM nomifensine, a competitive DA uptake inhibitor, was added. Even in the presence of nomifensine, 1 microM chlorpromazine had no effect on the DA concentration-response curve. The addition of DMPH4, an artificial cofactor for TH, completely blocked DA-induced inhibition of enzymatic activity. These data suggest that direct autoreceptor control of synaptosomal TH activity does not exist in vitro, and that DA-induced inhibition of TH occurs subsequent to reuptake via classical feedback inhibition, presumably by competitive displacement of the necessary endogenous cofactor.