Key Challenges and Recommendations for In Vitro Testing of Tobacco Products for Regulatory Applications: Consideration of Test Materials and Exposure Parameters.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and develop recommendations for optimal scientific and technical approaches for conducting in vitro assays, to assess potential toxicity within and across tobacco and various next generation nicotine and tobacco products (NGPs), including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDS). The third workshop (24-26 February 2020) summarised the key challenges and made recommendations concerning appropriate methods of test article generation and cell exposure from combustible cigarettes, HTPs and ENDS. Expert speakers provided their research, perspectives and recommendations for the three basic types of tobacco-related test articles: i) pad-collected material (PCM); ii) gas vapour phase (GVP); and iii) whole smoke/aerosol. These three types of samples can be tested individually, or the PCM and GVP can be combined. Whole smoke/aerosol can be bubbled through media or applied directly to cells at the air-liquid interface. Summaries of the speaker presentations and the recommendations developed by the workgroup are presented. Following discussion, the workshop concluded the following: that there needs to be greater standardisation in aerosol generation and collection processes; that methods for testing the NGPs need to be developed and/or optimised, since simply mirroring cigarette smoke testing approaches may be insufficient; that understanding and quantitating the applied dose is fundamental to the interpretation of data and conclusions from each study; and that whole smoke/aerosol approaches must be contextualised with regard to key information, including appropriate experimental controls, environmental conditioning, analytical monitoring, verification and performance criteria.

Evolutionary history of novel genes on the tammar wallaby Y chromosome: Implications for sex chromosome evolution.

We report here the isolation and sequencing of 10 Y-specific tammar wallaby (Macropus eugenii) BAC clones, revealing five hitherto undescribed tammar wallaby Y genes (in addition to the five genes already described) and several pseudogenes. Some genes on the wallaby Y display testis-specific expression, but most have low widespread expression. All have partners on the tammar X, along with homologs on the human X. Nonsynonymous and synonymous substitution ratios for nine of the tammar XY gene pairs indicate that they are each under purifying selection. All 10 were also identified as being on the Y in Tasmanian devil (Sarcophilus harrisii; a distantly related Australian marsupial); however, seven have been lost from the human Y. Maximum likelihood phylogenetic analyses of the wallaby YX genes, with respective homologs from other vertebrate representatives, revealed that three marsupial Y genes (HCFC1X/Y, MECP2X/Y, and HUWE1X/Y) were members of the ancestral therian pseudoautosomal region (PAR) at the time of the marsupial/eutherian split; three XY pairs (SOX3/SRY, RBMX/Y, and ATRX/Y) were isolated from each other before the marsupial/eutherian split, and the remaining three (RPL10X/Y, PHF6X/Y, and UBA1/UBE1Y) have a more complex evolutionary history. Thus, the small marsupial Y chromosome is surprisingly rich in ancient genes that are retained in at least Australian marsupials and evolved from testis-brain expressed genes on the X.

In vitro permeation of nicotine and tobacco specific nitrosamines from smokeless tobacco product extracts in a 3D buccal tissue model.

Tobacco product use is a risk factor in the development of oral cancer, although epidemiology studies show this risk is far less with smokeless tobacco product use than cigarette smoking. While smokeless tobacco contains harmful and potentially harmful constituents (HPHCs), the oral permeation of HPHCs in oral tobacco products is not completely understood. To improve the understanding, three different extract concentrations of the CORESTA reference products (CRP) for snus (CRP1.1) and moist snuff (CRP2.1) were applied to cellular tissue derived from two donors of EpiOral™ model, a 3D human buccal model, and permeation of nicotine and tobacco-specific nitrosamines (TSNAs) were measured over two hours. Permeation of 0.15% caffeine in complete artificial saliva and cell viability were also measured. Results showed that a consistent and concentration dependent cumulative permeation of nicotine and TSNAs was observed with high percent recovery in all conditions. A high degree of sensitivity was seen for all analytes, with minimal cytotoxicity for both CRPs. The data presented here show the EpiOral™ model is fit-for-purpose to evaluate the permeation of nicotine and TSNAs in nicotine-containing snus and moist snuff oral tobacco.

Evaluation of Cytotoxicity and Oxidative Stress of Whole Aerosol from Vuse Alto ENDS Products.

Assessment of in vitro cytotoxicity is an important component of tobacco product toxicological evaluations. However, current methods of regulatory testing involve exposing monolayer cell cultures to various preparations of aerosols from cigarettes or other emerging products such as electronic nicotine delivery systems (ENDS), which are not representative of human exposure. In the present study, a whole aerosol (WA) system was used to expose lung epithelial cultures (2D and 3D) to determine the potential of six Vuse Alto ENDS products that varied in nicotine content (1.8%, 2.4%, and 5%) and flavors (Golden Tobacco, Rich Tobacco, Menthol, and Mixed Berry), along with a marketed ENDS and a marked cigarette comparator to induce cytotoxicity and oxidative stress. The WA from the Vuse Alto ENDS products was not cytotoxic in the NRU and MTT assays, nor did it activate the Nrf2 reporter gene, a marker of oxidative stress. In summary, Vuse Alto ENDS products did not induce cytotoxic or oxidative stress responses in the in vitro models. The WA exposures used in the 3D in vitro models described herein may be better suited than 2D models for the determination of cytotoxicity and other in vitro functional endpoints and represent alternative models for regulatory evaluation of tobacco products.

In vitro toxicological evaluation of glo menthol and non-menthol heated tobacco products.

Heated tobacco products (HTPs) are non-combustible, inhaled tobacco products that generate an aerosol with fewer and lower levels of toxicants, with a potential to reduce risk relative to cigarette smoking. Here, we assessed in vitro toxicological effects of three menthol (glo neo neoCLICK, neo Smooth Menthol and Fresh Menthol) and one non-menthol (neo Smooth Tobacco) variants of glo HTP, along with market comparators for cigarettes and HTPs. Limited chemical characterization of the study products revealed significantly lower levels of acetaldehyde, acrolein, crotanaldehyde and formaldehyde in test samples from HTPs than those from cigarettes. The glo HTPs were non-mutagenic in the bacterial reverse mutagenesis assay. Although, the whole aerosol exposures of glo HTPs were classified as genotoxic in the in vitro micronucleus assay, and cytotoxic in the NRU (monolayer) and MTT (3 dimensional EpiAirway™ tissues) assays, the cigarette comparators were the most toxic study products in each of these assessments. Further, glo HTPs elicited oxidative stress responses only at the highest dose tested, whereas the cigarette comparators were potent inducers of oxidative stress at substantially lower doses in the EpiAirway tissues. The comparator (non-glo) HTP results were similar to the glo HTPs in these assays. Thus, the glo HTPs exhibit substantially lower toxicity compared to cigarettes.

Novel nicotinic acetylcholine receptor agonists containing carbonyl moiety as a hydrogen bond acceptor.

A novel series of α4ß2 nAChR agonists lacking common pyridine or its bioisosteric heterocycle have been disclosed. Essential pharmacophoric elements of the series are exocyclic carbonyl moiety as a hydrogen bond acceptor and secondary amino group within diaza- or azabicyclic scaffold. Computer modeling studies suggested that molecular shape of the ligand also contributes to promotion of agonism. Proof of concept for improving working memory performance in a novel object recognition task has been demonstrated on a representative of the series, 3-propionyl-3,7-diazabicyclo[3.3.0]octane (34).

A comparison of cigarette smoke test matrices and their responsiveness in the mouse lymphoma assay: A case study.

No cigarette smoke test matrix is without limitation, due to the complexity of the starting aerosol and phase to phase dynamics. It is impossible to capture all chemicals at the same level of efficiency, therefore, any test matrix will inadvertently or by design fractionate the test aerosol. This case study examines how four different test matrices derived from cigarette smoke can be directly compared. The test matrices assessed were as follows, total particulate matter (TPM), gas vapour phase (GVP), a combination of TPM + GVP and whole aerosol (WA). Here we use an example assay, the mouse lymphoma assay (MLA) to demonstrate that data generated across four cigarette smoke test matrices can be compared. The results show that all test matrices were able to induce positive mutational events, but with clear differences in the biological activity (both potency and toxicity) between them. TPM was deemed the most potent test article and by extension, the particulate phase is interpreted as the main driver of genotoxic induced responses in the MLA. However, the results highlight that the vapour phase is also active. MLA appeared responsive to WA, with potentially lower potency, compared to TPM approaches. However, this observation is caveated in that the WA approaches used for comparison were made on a newly developed experimental method using dose calculations. The TPM + GVP matrix had comparable activity to TPM alone, but interestingly induced a greater number of mutational events at comparable relative total growth (RTG) and TPM-equivalent doses when compared to other test matrices. In conclusion, this case study highlights the importance of understanding test matrices in response to the biological assay being assessed and we note that not all test matrices are equal.

Characterization of smoke and aerosol deliveries from combustible cigarettes, heated tobacco products and electronic nicotine delivery systems in the Vitrocell® Mammalian 6/48 exposure module.

The rapid development associated with Next Generation Tobacco Products (NGTP) has necessitated the development of high throughput methodologies to test their genotoxic potential in vitro when compared to conventional cigarette smoke (CS). An assessment of two Vitrocell® Mammalian 6/48 exposure modules in three independent experiments was made by comparing results from multiple dosimetric techniques applied to aerosol generated from 3R4F Kentucky Reference cigarettes, commercially available electronically heated tobacco product (eHTP) and Electronic Nicotine Delivery System (ENDS) using the Vitrocell® VC10®. Real-time aerosol particle concentration was assessed by means of light scattering photometers and expressed as area under the curve (∑AUC). Nicotine concentrations were determined analytically by LC/MS. Humectant amount and distribution was assessed for eHTP and ENDS by the quantification of free glycerol in a phosphate buffered saline (PBS) trap, whereas total particulate matter (TPM) was assessed in the 3R4F cigarettes by the fluorescence of the particulate at 485 nm in anhydrous dimethyl sulfoxide (DMSO) trap within the exposure. Dose was adjusted by means of the addition of ambient air to dilute the whole smoke/aerosol in L/min and sampled into the system at a rate of 5 mL/min. Dilution of CS ranged from 8.0 to 0.5 L/min and for the eHTP and ENDS ranged from 4 to 0 L/min (undiluted). Dosimetric analysis of the system showed good concordance within replicates (p-values ranged from p = 0.3762 to p = 0.8926) and showed that the Vitrocell® Mammalian 6/48 is a viable means for genotoxic assessment of aerosol generated from both conventional cigarettes and NGTP. Results demonstrate the need to tailor dosimetry approaches to different aerosols due to variations in the physio-chemical composition, with a multi-dosimetry approach recommended.

Therapeutic potential of novel selective drugs targeting nicotinic acetylcholine receptors.

The potential therapeutic benefit of nicotinic ligands in a variety of neurodegenerative pathologies involving the CNS has energized research efforts to develop nicotinic acetylcholine receptor (nAChR) subtype-selective ligands (Bencherif and Schmitt, 2005). In particular, there has been a concerted effort to develop nicotinic compounds with selectivity for CNS nAChRs as potential pharmaceutical tools in the management of these disorders. Clinical and experimental data demonstrate a central role for alpha7 and alpha4beta2 nAChRs in cognitive function, sensory processing, mood, and neuroprotection (Bencherif and Schmitt, 2005; Buccafusco et al., 2005). The development of safe alpha7-selective ligands has been hampered by their lack of discrimination with hERG channels and 5-HT3 receptors. We have developed a number of compounds that display nanomolar affinity to the alpha7 and/or the alpha4beta2 receptor. Investigation of alpha7 functional activity showed a full range of activities from antagonists to full agonists without any significant activity at the human 5-HT3 receptor, P450 isozymes, hERG channels, or in the AMES test. Our findings demonstrate that potent and highly selective nAChR ligands can be designed.

Dissociable effects of lidocaine inactivation of the rostral and caudal basolateral amygdala on the maintenance and reinstatement of cocaine-seeking behavior in rats.

Cocaine addiction is a chronically relapsing brain disease, but its neural basis is not yet well understood. Clinical reports underscore the possible importance of associative processes for regulating at least some aspects of cocaine addiction. The present study reports the effects of reversible lidocaine-induced inactivation of rostral basolateral amygdala (rBLA) and caudal basolateral amygdala (cBLA) regions on the maintenance and reinstatement of drug-seeking behavior in rats trained to self-administer 1 mg/kg cocaine under a second order schedule of drug delivery. Both regions of the basolateral amygdala were investigated because they have dissociable effects on cognitive task performance. Results demonstrated that after self-administration training and a period of extinction and abstinence, lidocaine inactivation of the rBLA and cBLA attenuated the reinstatement of drug-seeking behavior induced by cocaine-associated cues examined in conjunction with a single priming injection of cocaine. In contrast, lidocaine inactivation of only the rBLA blocked reinstatement of drug-seeking behavior induced by cocaine-associated cues examined alone. Additional differences were shown during cocaine maintenance testing where inactivation of only the cBLA attenuated drug-seeking behavior. Drug intake was not altered. Thus, the rBLA and cBLA appear to selectively and dissociably regulate drug-seeking behavior under conditions of cocaine abstinence (cue-induced reinstatement) and repeated cocaine use (maintenance), respectively. These findings suggest that the basolateral amygdala may be more functionally heterogeneous than commonly thought for regulating drug-seeking behavior. The basis for this dissociation might be related to neuroanatomical connections of the rBLA and cBLA with segregated, but parallel, corticostriatalpallidothalamic circuits.

KRAS Genotype Correlates with Proteasome Inhibitor Ixazomib Activity in Preclinical In Vivo Models of Colon and Non-Small Cell Lung Cancer: Potential Role of Tumor Metabolism.

In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition.

Complementary tasks to measure working memory in distinct prefrontal cortex subregions in rats.

Acquisition of odor-guided or visually-guided delayed win-shift behavior was evaluated in rats after lidocaine-induced inactivation within the agranular insular area of the prefrontal cortex (PFC) or the prelimbic area of the PFC. Additional sites and tasks were used to control for neuroanatomical and behavioral specificity of lidocaine inactivation of the agranular insular and prelimbic areas. Results showed that acquisition of the odor-guided delayed win-shift task was dependent on the agranular insular area, whereas acquisition of the visually-guided version was dependent on the prelimbic area. This dissociation suggests that the stimulus modality used is critical for revealing working memory functions of different PFC subregions. The described methods provide a complementary means to study working memory in PFC subregions using a radial-arm maze.

Characterization of the discriminative stimulus effects of N-methyl- D-aspartate ligands under different ethanol training conditions in the cynomolgus monkey ( Macaca fascicularis).

RATIONALE: The current study was designed to extend our knowledge of the N-methyl- D-aspartate (NMDA) glutamate receptor system in mediating the discriminative stimulus effects of ethanol in non-human primates. OBJECTIVES: To characterize the discriminative stimulus effects of the NMDA uncompetitive antagonists dizocilpine, phencyclidine (PCP) and ketamine in male and female monkeys under different ethanol training conditions. METHODS: Adult male ( n=8) and female ( n=9) cynomolgus monkeys ( Macaca fascicularis) were divided into four groups and trained to discriminate 1.0 g/kg ethanol ( n=8) versus water or 2.0 g/kg ethanol ( n=9) versus water in a 2 x 2 design with training dose and sex as main group factors. Ethanol (20% w/v) solutions were administered intragastrically (IG) and responding was maintained under a fixed ratio schedule of food reinforcement. Dose-response determinations for dizocilpine [IG and intramuscular (IM)], PCP (IM) and ketamine (IM) were made under two training intervals (30 and 60 min). RESULTS: Dizocilpine, PCP and ketamine dose-dependently substituted for ethanol in three of four training conditions, the notable exception being in males trained with 2.0 g/kg ethanol. Ethanol-like discriminative stimulus effects were greater with IM dizocilpine than with IG dizocilpine. At the lower ethanol training dose (1.0 g/kg), there were no sex differences in the ethanol-like discriminative stimulus effects of dizocilpine, PCP or ketamine, nor were there sex differences in the potencies to produce ethanol-like discriminative stimulus effects. Sex differences were readily apparent with the higher ethanol training dose (2.0 g/kg), with the NMDA ligands failing to substitute for ethanol in male monkeys, probably due to the rate-suppressive effects of these compounds. CONCLUSIONS: These data suggest that NMDA receptor-mediated activity is a component to the discriminative stimulus effects of ethanol in male and female nonhuman primates. However, NMDA uncompetitive antagonists were less likely to produce discriminative stimulus effects similar to a high ethanol training dose in male monkeys. In comparison to consistent substitution by GABA(A) positive modulators for ethanol, substitution patterns produced by NMDA uncompetitive antagonists suggest a less robust mediation of the ethanol discriminative stimulus through NMDA receptor systems in nonhuman primates.

Stimulus-response functions of the lateral dorsal striatum and regulation of behavior studied in a cocaine maintenance/cue reinstatement model in rats.

RATIONALE: To investigate potential neurocognitive mechanisms underlying drug-seeking and drug-taking behavior, the effects of reversible lidocaine-induced inactivation of the lateral dorsal striatum (DST) on behavior studied in a drug maintenance/cue reinstatement model were evaluated. This region of the DST was investigated because it selectively regulates stimulus-response learning that is disrupted by 10 microg of bilaterally infused lidocaine. METHODS: Rats ( n=6) were trained to self-administer 1 mg/kg per infusion cocaine under a second-order schedule of drug delivery. The effects of bilateral lidocaine (30-100 microg) inactivation of the lateral DST were evaluated during drug maintenance tests as well as during tests in which responding was reinstated by cocaine-associated cues presented in combination with a cocaine priming injection. The lower 10 microg dose was used to examine the effects of lidocaine on reinstatement of responding induced by presentation of cues alone. RESULTS: During drug maintenance tests, drug-seeking behavior was significantly increased after inactivation by 100 microg lidocaine. The number of infusions earned did not change. During cue-induced reinstatement tests preceded by a cocaine priming injection, 100 microg lidocaine significantly decreased both drug-seeking behavior and the number of infusion-paired light deliveries earned. During reinstatement tests with cues presented alone, inactivation of the lateral DST by 10 microg lidocaine did not influence either behavior. CONCLUSIONS: These findings suggest that stimulus-response functions of the lateral DST may regulate the dose-related effects of self-administered cocaine because the lidocaine-induced changes in behavior during the maintenance and cocaine priming tests resembled the effects of exposure to increasingly lower doses of cocaine, respectively. Given the lack of an effect of lidocaine during the cues-alone tests, the lateral DST does not appear to regulate drug-seeking behavior per se (i.e., responding maintained by drug-associated cues at times when drug is not available).

Hippocampal memory system function and the regulation of cocaine self-administration behavior in rats.

There is considerable interest in elucidating neurocognitive mechanisms of cocaine addiction. This report focuses on the hippocampal memory system. Using food reward, two cognitive tasks were examined after lidocaine inactivation of the dorsal (dSUB) or ventral (vSUB) subiculum, the primary hippocampal output regions in rats. These tasks were conducted to first identify functionally relevant stereotaxic coordinates within the hippocampal memory system, in order to then examine its role in regulating drug-seeking and drug-taking behavior studied under a contextually discriminable FI 5-min(FR5:S) second-order schedule of cocaine and brief stimulus delivery. Inactivation of the dSUB and vSUB with 10microg lidocaine impaired hippocampal-dependent win-shift performance. Amygdalar-dependant conditioned cue preference, used as a test for behavioral specificity of lidocaine, was not affected following inactivation of either site. Inactivation of the dSUB with 100 microg lidocaine significantly reduced drug-seeking and drug-taking behavior studied during the cocaine self-administration maintenance phase. Following extinction, inactivation of neither the dSUB nor vSUB influenced reinstatement of drug-seeking behavior induced by drug-paired cues presented alone or with a cocaine priming injection. The impairments in win-shift performance are consistent with the spatial processing functions of the dSUB and vSUB, and the reduction in drug-taking behavior is consistent with the non-spatial temporal processing functions of the dSUB. The lack of an effect of dSUB and vSUB inactivation on reinstatement of drug-seeking behavior may relate to the fact that the contextual associations with cocaine were well-practiced at the time of cue reinstatement testing, and therefore, drug-seeking behavior was likely regulated by nonhippocampal-dependent mechanisms.

α4ß2 Nicotinic receptors play a role in the nAChR-mediated decline in L-dopa-induced dyskinesias in parkinsonian rats.

L-Dopa-induced dyskinesias are a serious long-term side effect of dopamine replacement therapy for Parkinson's disease for which there are few treatment options. Our previous studies showed that nicotine decreased l-dopa-induced abnormal involuntary movements (AIMs). Subsequent work with knockout mice demonstrated that α6ß2* nicotinic receptors (nAChRs) play a key role. The present experiments were done to determine if α4ß2* nAChRs are also involved in l-dopa-induced dyskinesias. To approach this, we took advantage of the finding that α6ß2* nAChRs are predominantly present on striatal dopaminergic nerve terminals, while a significant population of α4ß2* nAChRs are located on other neurons. Thus, a severe dopaminergic lesion would cause a major loss in α6ß2*, but not α4ß2* nAChRs. Experiments were therefore done in which rats were unilaterally lesioned with 6-hydroxydopamine, at a dose that led to severe nigrostriatal damage. The dopamine transporter, a dopamine nerve terminal marker, was decreased by >99%. This lesion also decreased striatal α6ß2* nAChRs by 97%, while α4ß2* nAChRs were reduced by only 12% compared to control. A series of ß2* nAChR compounds, including TC-2696, TI-10165, TC-8831, TC-10600 and sazetidine reduced l-dopa-induced AIMs in these rats by 23-32%. TC-2696, TI-10165, TC-8831 were also tested for parkinsonism, with no effect on this behavior. Tolerance did not develop with up to 3 months of treatment. Since α4α5ß2 nAChRs are also predominantly on striatal dopamine terminals, these data suggest that drugs targeting α4ß2 nAChRs may reduce l-dopa-induced dyskinesias in late stage Parkinson's disease.

Neuronal nicotinic receptor agonists ameliorate spontaneous motor asymmetries and motor discoordination in a unilateral mouse model of Parkinson's disease.

The degeneration of the nigrostriatal dopamine (DA) system underlies the motor deficits in Parkinson's disease (PD). In recent years, epidemiological reports that smokers have lower incidences of PD have brought attention to the nicotinic acetylcholine system as a potential target for novel therapeutics. Nicotine, an agonist of neuronal nicotinic receptors (NNRs), modulates functions relevant to PD via stimulation of dopaminergic transmission in the nigrostriatal pathway, particularly via activation of α6ß2* and α4ß2* NNRs. Recently, reduced support of DA neurons by neurotrophic growth factors has been described in PD. Fibroblast growth factor (FGF) is critical for the development and protection of adult DA neurons. In FGF-2 knockout mice and the related th-fgfr1(tk-) mouse model there is heightened sensitivity to DA neuronal oxidative neurotoxin 6-hydroxydopamine (6-OHDA). In the present study, FGF-deficient transgenic mice th-fgfr1(tk-) were used to analyze the effects of novel full (TC-8831) and partial (TC-8581) agonists of ß2-containing nicotinic receptors on impaired motor behavior following unilateral 6-OHDA lesions. The lesions generated spontaneous (drug-naïve) turning asymmetries that correlated exponentially with the depletion of DA biomarkers in the lesioned striata. These mice also exhibited a reduced capacity to remain on the accelerating rotarod. Oral administration of TC-8831, an NNR agonist with high specificity for ß2 subunits and a full agonist at producing DA release from striatal synaptosomes, attenuated unidirectional turning and improved motor coordination. In contrast, partial ß2 NNR agonist TC-8581 had no effect on behaviors in this model. This study demonstrates the potential of NNR targeting-compounds to facilitate motor function in PD.

TC-8831, a nicotinic acetylcholine receptor agonist, reduces L-DOPA-induced dyskinesia in the MPTP macaque.

Long-term L-DOPA treatment for Parkinson's disease (PD) is limited by motor complications, particularly L-DOPA-induced dyskinesia (LID). A therapy with the ability to ameliorate LID without reducing anti-parkinsonian benefit would be of great value. We assessed the ability of TC-8831, an agonist at nicotinic acetylcholine receptors (nAChR) containing α6ß2/α4ß2 subunit combinations, to provide such benefits in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- (MPTP) lesioned macaques with established LID. Animals were treated orally for consecutive 14-day periods with twice-daily vehicle (weeks 1-2) or TC-8831 (0.03, 0.1 or 0.3 mg/kg, weeks 3-8). L-DOPA was also administered, once-daily, (weeks 1-12, median-dose 30 mg/kg, p.o.). For the following two-weeks (weeks 9-10), TC-8831 was washed out, while once-daily L-DOPA treatment was maintained. The effects of once-daily amantadine (3 mg/kg, p.o.) were then assessed over weeks 11-12. LID, parkinsonism, duration and quality of ON-time were assessed weekly by a neurologist blinded to treatment. TC-8831 reduced the duration of 'bad' ON-time (ON-time with disabling dyskinesia) by up to 62% and decreased LID severity (median score 18 cf. 34 (vehicle), 0.1 mg/kg, 1-3 h period). TC-8831 also significantly reduced choreiform and dystonic dyskinesia (median scores 6 and 31 cf. 19 and 31 respectively (vehicle), both 0.03 mg/kg, 1-3 h). At no time did TC-8831 treatment result in a reduction in anti-parkinsonian benefit of L-DOPA. By comparison, amantadine also significantly reduced dyskinesia and decreased 'bad' ON-time (up to 61%) but at the expense of total ON-time (reduced by up to 23%). TC-8831 displayed robust anti-dyskinetic actions and improved the quality of ON-time evoked by L-DOPA without any reduction in anti-parkinsonian benefit.