Pharmacological Characterization of Levorphanol, a G-Protein Biased Opioid Analgesic.

BACKGROUND: Levorphanol is a potent analgesic that has been used for decades. Most commonly used for acute and cancer pain, it also is effective against neuropathic pain. The recent appreciation of the importance of functional bias and the uncovering of multiple µ opioid receptor splice variants may help explain the variability of patient responses to different opioid drugs. METHODS: Here, we evaluate levorphanol in a variety of traditional in vitro receptor binding and functional assays. In vivo analgesia studies using the radiant heat tail flick assay explored the receptor selectivity of the responses through the use of knockout (KO) mice, selective antagonists, and viral rescue approaches. RESULTS: Receptor binding studies revealed high levorphanol affinity for all the µ, δ, and κ opioid receptors. In S-GTPγS binding assays, it was a full agonist at most µ receptor subtypes, with the exception of MOR-1O, but displayed little activity in ß-arrestin2 recruitment assays, indicating a preference for G-protein transduction mechanisms. A KO mouse and selective antagonists confirmed that levorphanol analgesia was mediated through classical µ receptors, but there was a contribution from 6 transmembrane targets, as illustrated by a lower response in an exon 11 KO mouse and its rescue with a virally transfected 6 transmembrane receptor splice variant. Compared to morphine, levorphanol had less respiratory depression at equianalgesic doses. CONCLUSIONS: While levorphanol shares many of the same properties as the classic opioid morphine, it displays subtle differences that may prove helpful in its clinical use. Its G-protein signaling bias is consistent with its diminished respiratory depression, while its incomplete cross tolerance with morphine suggests it may prove valuable clinically with opioid rotation.

Genetic dissociation of morphine analgesia from hyperalgesia in mice.

RATIONALE: Morphine is the prototypic mu opioid, producing its analgesic actions through traditional 7 transmembrane domain (7TM) G-protein-coupled receptors generated by the mu opioid receptor gene (Oprm1). However, the Oprm1 gene undergoes extensive alternative splicing to yield three structurally distinct sets of splice variants. In addition to the full-length 7TM receptors, it produces a set of truncated variants comprised of only 6 transmembrane domains (6TM). OBJECTIVES: This study explored the relative contributions of 7TM and 6TM variants in a range of morphine actions. METHODS: Groups of male and mixed-gender wild-type and exon 11 Oprm1 knockout mice were examined in a series of behavioral assays measuring analgesia, hyperalgesia, respiration, and reward in conditioned place preference assays. RESULTS: Loss of the 6TM variants in an exon 11 knockout (E11 KO) mouse did not affect morphine analgesia, reward, or respiratory depression. However, E11 KO mice lacking 6TM variants failed to show morphine-induced hyperalgesia, developed tolerance more slowly than wild-type mice, and did not display hyperlocomotion. CONCLUSIONS: Together, our findings confirm the established role of 7TM mu receptor variants in morphine analgesia, reward, and respiratory depression, but reveal an unexpected obligatory role for 6TM variants in morphine-induced hyperalgesia and a modulatory role in morphine tolerance and dependence.

Tetrapeptide Endomorphin Analogs Require Both Full Length and Truncated Splice Variants of the Mu Opioid Receptor Gene Oprm1 for Analgesia.

The mu opioid receptor gene undergoes extensive alternative splicing. Mu opioids can be divided into three classes based on the role of different groups of splice variants. Morphine and methadone require only full length seven transmembrane (7TM) variants for analgesia, whereas IBNtxA (3'-iodobenzyol-6ß-naltrexamide) needs only truncated 6TM variants. A set of endomorphin analogs fall into a third group that requires both 6TM and 7TM splice variants. Unlike morphine, endomorphin 1 and 2, DAPP (Dmt,d-Ala-Phe-Phe-NH2), and IDAPP (3'-iodo-Dmt-d-Ala-Phe-Phe-NH2) analgesia was lost in an exon 11 knockout mouse lacking 6TM variants. Restoring 6TM variant expression in a knockout mouse lacking both 6TM and 7TM variants failed to rescue DAPP or IDAPP analgesia. However, re-establishing 6TM expression in an exon 11 knockout mouse that still expressed 7TM variants did rescue the response, consistent with the need for both 6TM and 7TM variants. In receptor binding assays, 125I-IDAPP labeled more sites (Bmax) than 3H-DAMGO ([d-Ala2,N-MePhe4,Gly(ol)5]-enkephalin) in wild-type mice. In exon 11 knockout mice, 125I-IDAPP binding was lowered to levels similar to 3H-DAMGO, which remained relatively unchanged compared to wild-type mice. 125I-IDAPP binding was totally lost in an exon 1/exon 11 knockout model lacking all Oprm1 variant expression, confirming that the drug was not cross labeling non-mu opioid receptors. These findings suggested that 125I-IDAPP labeled two populations of mu binding sites in wild-type mice, one corresponding to 7TM variants and the second dependent upon 6TM variants. Together, these data indicate that endomorphin analogs represent a unique, genetically defined, and distinct class of mu opioid analgesic.

Mitragynine/Corynantheidine Pseudoindoxyls As Opioid Analgesics with Mu Agonism and Delta Antagonism, Which Do Not Recruit ß-Arrestin-2.

Natural products found in Mitragyna speciosa, commonly known as kratom, represent diverse scaffolds (indole, indolenine, and spiro pseudoindoxyl) with opioid activity, providing opportunities to better understand opioid pharmacology. Herein, we report the pharmacology and SAR studies both in vitro and in vivo of mitragynine pseudoindoxyl (3), an oxidative rearrangement product of the corynanthe alkaloid mitragynine. 3 and its corresponding corynantheidine analogs show promise as potent analgesics with a mechanism of action that includes mu opioid receptor agonism/delta opioid receptor antagonism. In vitro, 3 and its analogs were potent agonists in [(35)S]GTPγS assays at the mu opioid receptor but failed to recruit ß-arrestin-2, which is associated with opioid side effects. Additionally, 3 developed analgesic tolerance more slowly than morphine, showed limited physical dependence, respiratory depression, constipation, and displayed no reward or aversion in CPP/CPA assays, suggesting that analogs might represent a promising new generation of novel pain relievers.

Mediation of buprenorphine analgesia by a combination of traditional and truncated mu opioid receptor splice variants.

Buprenorphine has long been classified as a mu analgesic, although its high affinity for other opioid receptor classes and the orphanin FQ/nociceptin ORL1 receptor may contribute to its other actions. The current studies confirmed a mu mechanism for buprenorphine analgesia, implicating several subsets of mu receptor splice variants. Buprenorphine analgesia depended on the expression of both exon 1-associated traditional full length 7 transmembrane (7TM) and exon 11-associated truncated 6 transmembrane (6TM) MOR-1 variants. In genetic models, disruption of delta, kappa1 or ORL1 receptors had no impact on buprenorphine analgesia, while loss of the traditional 7TM MOR-1 variants in an exon 1 knockout (KO) mouse markedly lowered buprenorphine analgesia. Loss of the truncated 6TM variants in an exon 11 KO mouse totally eliminated buprenorphine analgesia. In distinction to analgesia, the inhibition of gastrointestinal transit and stimulation of locomotor activity were independent of truncated 6TM variants. Restoring expression of a 6TM variant with a lentivirus rescued buprenorphine analgesia in an exon 11 KO mouse that still expressed the 7TM variants. Despite a potent and robust stimulation of (35) S-GTPγS binding in MOR-1 expressing CHO cells, buprenorphine failed to recruit ß-arrestin-2 binding at doses as high as 10 µM. Buprenorphine was an antagonist in DOR-1 expressing cells and an inverse agonist in KOR-1 cells. Buprenorphine analgesia is complex and requires multiple mu receptor splice variant classes but other actions may involve alternative receptors.

Truncated mu opioid GPCR variant involvement in opioid-dependent and opioid-independent pain modulatory systems within the CNS.

The clinical management of severe pain depends heavily on opioids acting through mu opioid receptors encoded by the Oprm1 gene, which undergoes extensive alternative splicing. In addition to generating a series of prototypic seven transmembrane domain (7TM) G protein-coupled receptors (GPCRs), Oprm1 also produces a set of truncated splice variants containing only six transmembrane domains (6TM) through which selected opioids such as IBNtxA (3'-iodobenzoyl-6ß-naltrexamide) mediate a potent analgesia without many undesirable effects. Although morphine analgesia is independent of these 6TM mu receptor isoforms, we now show that the selective loss of the 6TM variants in a knockout model eliminates the analgesic actions of delta and kappa opioids and of α2-adrenergic compounds, but not cannabinoid, neurotensin, or muscarinic drugs. These observations were confirmed by using antisense paradigms. Despite their role in analgesia, loss of the 6TM variants were not involved with delta opioid-induced seizure activity, aversion to the kappa drug U50, 488H, or α2-mediated hypolocomotion. These observations support the existence of parallel opioid and nonopioid pain modulatory systems and highlight the ability to dissociate unwanted delta, kappa1, and α2 actions from analgesia.

Synthesis and characterization of a dual kappa-delta opioid receptor agonist analgesic blocking cocaine reward behavior.

3-Iodobenzoyl naltrexamine (IBNtxA) is a potent analgesic belonging to the pharmacologically diverse 6ß-amidoepoxymorphinan group of opioids. We present the synthesis and pharmacological evaluation of five analogs of IBNtxA. The scaffold of IBNtxA was modified by removing the 14-hydroxy group, incorporating a 7,8 double bond and various N-17 alkyl substituents. The structural modifications resulted in analogs with picomolar affinities for opioid receptors. The lead compound (MP1104) was found to exhibit approximately 15-fold greater antinociceptive potency (ED50 = 0.33 mg/kg) compared with morphine, mediated through the activation of kappa- and delta-opioid receptors. Despite its kappa agonism, this lead derivative did not cause place aversion or preference in mice in a place-conditioning assay, even at doses 3 times the analgesic ED50. However, pretreatment with the lead compound prevented the reward behavior associated with cocaine in a conditioned place preference assay. Together, these results suggest the promise of dual acting kappa- and delta-opioid receptor agonists as analgesics and treatments for cocaine addiction.

Radioligand Binding Assay for an Exon 11-Associated Mu Opioid Receptor Target.

Receptor binding provides a valuable approach for characterization of drugs and their receptors. There are three major families of opioid receptors: mu, delta, and kappa. Highly selective radioligands are available for all three classes of traditional receptors. Of the three, the mu receptor undergoes extensive alternative splicing, generating a number of traditional mu receptor subtypes as well as a nontraditional, truncated set of variants associated with exon 11. These exon 11-associated truncated variants are not readily labeled with current radioligands. Here we describe the synthesis of a radioiodinated ligand suitable for carrying out binding studies for this target.

Synthesis of Carfentanil Amide Opioids Using the Ugi Multicomponent Reaction.

We report a novel approach to synthesize carfentanil amide analogues utilizing the isocyanide-based four-component Ugi multicomponent reaction. A small library of bis-amide analogues of carfentanil was created using N-alkylpiperidones, aniline, propionic acid, and various aliphatic isocyanides. Our lead compound showed high affinity for mu (MOR) and delta opioid receptors (DOR) with no appreciable affinity for kappa (KOR) receptors in radioligand binding assays. The compound was found to be a mixed MOR agonist/partial DOR agonist in [(35)S]GTPγS functional assays, and it showed moderate analgesic potency in vivo. The compound showed no visible signs of physical dependence or constipation in mice. In addition, it produced less respiratory depression than morphine. Most mixed MOR/DOR opioids reported in the literature are peptides and thereby systemically inactive. Our approach utilizing a multicomponent reaction has the promise to deliver potent and efficacious small-molecule analgesics with potential clinical utility.

Synthesis and pharmacology of halogenated δ-opioid-selective [d-Ala(2)]deltorphin II peptide analogues.

Deltorphins are naturally occurring peptides produced by the skin of the giant monkey frog (Phyllomedusa bicolor). They are δ-opioid receptor-selective agonists. Herein, we report the design and synthesis of a peptide, Tyr-d-Ala-(pI)Phe-Glu-Ile-Ile-Gly-NH2 3 (GATE3-8), based on the [d-Ala(2)]deltorphin II template, which is δ-selective in in vitro radioligand binding assays over the µ- and κ-opioid receptors. It is a full agonist in [(35)S]GTPγS functional assays and analgesic when administered supraspinally to mice. Analgesia of 3 (GATE3-8) is blocked by the selective δ receptor antagonist naltrindole, indicating that the analgesic action of 3 is mediated by the δ-opioid receptor. We have established a radioligand in which (125)I is incorporated into 3 (GATE3-8). The radioligand has a KD of 0.1 nM in Chinese hamster ovary (CHO) cells expressing the δ receptor. Additionally, a series of peptides based on 3 (GATE3-8) was synthesized by incorporating various halogens in the para position on the aromatic ring of Phe(3). The peptides were characterized for binding affinity at the µ-, δ-, and κ-opioid receptors, which showed a linear correlation between binding affinity and the size of the halogen substituent. These peptides may be interesting tools for probing δ-opioid receptor pharmacology.

Nonlinear pharmacokinetics of (+/-)3,4-methylenedioxymethamphetamine (MDMA) and its pharmacodynamic consequences in the rat.

3,4-Methylenedioxymethamphetamine (MDMA) is a widely abused illicit drug that can cause severe and even fatal adverse effects. However, interest remains for its possible clinical applications in posttraumatic stress disorder and anxiety treatment. Preclinical studies to determine MDMA's safety are needed. We evaluated MDMA's pharmacokinetics and metabolism in male rats receiving 2.5, 5, and 10 mg/kg s.c. MDMA, and the associated pharmacodynamic consequences. Blood was collected via jugular catheter at 0, 0.5, 1, 2, 4, 6, 8, 16, and 24 hours, with simultaneous serotonin (5-HT) behavioral syndrome and core temperature monitoring. Plasma specimens were analyzed for MDMA and the metabolites (±)-3,4-dihydroxymethamphetamine (HHMA), (±)-4-hydroxy-3-methoxymethamphetamine (HMMA), and (±)-3,4-methylenedioxyamphetamine (MDA) by liquid chromatography-tandem mass spectrometry. After 2.5 mg/kg MDMA, mean MDMA Cmax was 164 ± 47.1 ng/ml, HHMA and HMMA were major metabolites, and <20% of MDMA was metabolized to MDA. After 5- and 10-mg/kg doses, MDMA areas under the curve (AUCs) were 3- and 10-fold greater than those after 2.5 mg/kg; HHMA and HMMA AUC values were relatively constant across doses; and MDA AUC values were greater than dose-proportional. Our data provide decisive in vivo evidence that MDMA and MDA display nonlinear accumulation via metabolic autoinhibition in the rat. Importantly, 5-HT syndrome severity correlated with MDMA concentrations (r = 0.8083; P < 0.0001) and core temperature correlated with MDA concentrations (r = 0.7595; P < 0.0001), suggesting that MDMA's behavioral and hyperthermic effects may involve distinct mechanisms. Given key similarities between MDMA pharmacokinetics in rats and humans, data from rats can be useful when provided at clinically relevant doses.

Stress differentially alters mu opioid receptor density and trafficking in parvalbumin-containing interneurons in the female and male rat hippocampus.

Stress differentially affects hippocampal-dependent learning relevant to addiction and morphology in male and female rats. Mu opioid receptors (MORs), which are located in parvalbumin (PARV)-containing GABAergic interneurons and are trafficked in response to changes in the hormonal environment, play a critical role in promoting principal cell excitability and long-term potentiation. Here, we compared the effects of acute and chronic immobilization stress (AIS and CIS) on MOR trafficking in PARV-containing neurons in the hilus of the dentate gyrus in female and male rats using dual label immunoelectron microscopy. Following AIS, the density of MOR silver-intensified gold particles (SIGs) in the cytoplasm of PARV-labeled dendrites was significantly reduced in females (estrus stage). Conversely, AIS significantly increased the proportion of cytoplasmic MOR SIGs in PARV-labeled dendrites in male rats. CIS significantly reduced the number of PARV-labeled neurons in the dentate hilus of males but not females. However, MOR/PARV-labeled dendrites and terminals were significantly smaller in CIS females, but not males, compared with controls. Following CIS, the density of cytoplasmic MOR SIGs increased in PARV-labeled dendrites and terminals in females. Moreover, the proportion of near-plasmalemmal MOR SIGs relative to total decreased in large PARV-labeled dendrites in females. After CIS, no changes in the density or trafficking of MOR SIGs were seen in PARV-labeled dendrites or terminals in males. These data show that AIS and CIS differentially affect available MOR pools in PARV-containing interneurons in female and male rats. Furthermore, they suggest that CIS could affect principal cell excitability in a manner that maintains learning processes in females but not males.

Oral fluid nicotine markers to assess smoking status and recency of use.

INTRODUCTION: Oral fluid collection is noninvasive and easily observed making it an attractive matrix for objectively determining smoking status. Despite large intersubject variability, cotinine oral fluid concentrations correlate with cigarettes smoked per day (CPD). Few studies, however, assessed nicotine markers in oral fluid other than cotinine; other markers might improve smoking status assessment and/or time of last cigarette. MATERIALS AND METHODS: Smoking histories and oral fluid specimens were collected from nontreatment-seeking light (1-10 CPD) and heavy smokers (greater than 10 CPD) and from environmentally exposed and nonexposed nonsmokers who provided written informed consent for this Institutional Review Board-approved study. Nicotine, cotinine, hydroxycotinine (OH-cotinine), and norcotinine oral fluid concentrations were quantified by liquid chromatography tandem mass spectrometry. RESULTS: Comparison of 1, 3, and 10 ng/mL oral fluid liquid chromatography tandem mass spectrometry cutoffs demonstrated that 10-ng/mL cutoffs performed optimally for cotinine, OH-cotinine, nicotine, and norcotinine identifying 98%, 97%, 88%, and 15% of self-reported smokers; 1% nonsmokers had greater than 10 ng/mL cotinine. No self-reported nonsmoker had greater than 10 ng/mL OH-cotinine, nicotine, or norcotinine. Norcotinine was only identified in smokers' oral fluid. Oral fluid nicotine, cotinine, and nicotine/cotinine ratios were correlated with time of last smoking (r = -0.53, -0.23, and -0.51; P < 0.05) and CPD (r = 0.35, 0.26, and 0.33; P < 0.01), respectively. DISCUSSION AND CONCLUSION: OH-cotinine performed slightly better than cotinine for distinguishing smokers from nonsmokers and should be considered as an additional oral fluid smoking indicator. Further research is required to determine if oral fluid norcotinine is a marker for distinguishing light and heavy smokers. Moderate correlations suggest nicotine, cotinine, and nicotine/cotinine ratios may be useful for determining smoking recency in "spot samples" collected during nicotine cessation treatment.

Relative performance of common biochemical indicators in detecting cigarette smoking.

AIMS: Many cities have banned indoor smoking in public places. Thus, an updated recommendation for a breath carbon monoxide (CO) cut-off is needed that optimally determines smoking status. We evaluated and compared the performance of breath CO and semiquantitative cotinine immunoassay test strips (urine and saliva NicAlert®) alone and in combination. DESIGN: Cross-sectional study. SETTING: Urban drug addiction research and treatment facility. PARTICIPANTS: Ninety non-treatment-seeking smokers and 82 non-smokers. MEASUREMENTS: Participants completed smoking histories and provided breath CO, urine and saliva specimens. Urine and saliva specimens were assayed for cotinine by NicAlert® and liquid chromatography-tandem mass spectrometry (LCMSMS). FINDINGS: An optimal breath CO cut-off was established using self-report and LCMSMS analysis of cotinine, an objective indicator, as reference measures. Performance of smoking indicators and combinations were compared to the reference measures. Breath CO ≥5 parts per million (p.p.m.) optimally discriminated smokers from non-smokers. Saliva NicAlert® performance was less effective than the other indicators. CONCLUSIONS: In surveys of smokers and non-smokers in areas with strong smoke-free laws, the breath carbon monoxide cut-off that discriminates most effectively appears to be ≥5 p.p.m. rather than the ≥10 p.p.m. cut-off often used. These findings may not generalize to clinical trials, regions with different carbon monoxide pollution levels or areas with less stringent smoke-free laws.

Incubation of cue-induced cigarette craving during abstinence in human smokers.

BACKGROUND: Abstinent drug users remain at risk for relapse long after withdrawal subsides. Animal studies indicate that responses to drug-related cues not only persist but increase with abstinence, a phenomenon termed "incubation of drug craving." It is unknown whether cue-induced craving increases, decreases, or remains constant with abstinence in humans. We investigated effects of abstinence on cue-induced craving in cigarette smokers. METHODS: Eighty-six non-treatment-seeking, adult smokers (≥10 cigarettes daily) were paid to abstain for 7 (Group 1), 14 (Group 2), or 35 (Groups 3 and 4) days. Abstinence was verified daily. Groups 1, 2, and 3 underwent a single cue session on the final abstinence day (7, 14, or 35). Group 4 viewed cues on Days 7, 14, and 35. RESULTS: Between and within groups, smoking-cue-induced craving increased with abstinence on some measures. Cue-induced craving was greater in Group 3 (35-day) compared with Group 1 (7-day). Within Group 4, cue-induced craving was greater at 35 than 14 days. Cue-induced craving did not decrease with abstinence on any measure. CONCLUSIONS: We present initial evidence of incubation of cue-induced craving in humans. The observation that cue-induced craving increases with abstinence, even as "background" craving and withdrawal symptoms subside, might have treatment implications.

Amphetamine analogs methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA) differentially affect speech.

RATIONALE: Most reports of the effects of methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA) on speech have been anecdotal. OBJECTIVES: The current study used a within-participant design to assess the effects of methamphetamine and MDMA on speech. MATERIALS AND METHODS: Eleven recreational users of amphetamines completed this inpatient, within-participant, double-blind study, during which they received placebo, methamphetamine (20, 40 mg), and MDMA (100 mg) on separate days. Following drug administration, study participants described movies viewed the previous evening and completed mood scales. RESULTS: Methamphetamine increased quantity of speech, fluency, and self-ratings of talkativeness and alertness, while it decreased the average duration of nonjuncture unfilled pauses. MDMA decreased fluency and increased self-ratings of inability to concentrate. To determine if methamphetamine- and MDMA-related effects were perceptible, undergraduates listened to the participants' movie descriptions and rated their coherence and the speaker's mood. Following methamphetamine, descriptions were judged to be more coherent and focused than they were following MDMA. CONCLUSIONS: Methamphetamine improved verbal fluency and MDMA adversely affected fluency. This pattern of effects is consistent with the effects of these drugs on functioning in other cognitive domains. In general, methamphetamine effects on speech were inconsistent with effects popularly attributed to this drug, while MDMA-related effects were in agreement with some anecdotal reports and discordant with others.

Tobacco, cocaine, and heroin: Craving and use during daily life.

BACKGROUND: Relationships among tobacco smoking, tobacco craving, and other drug use and craving may have treatment implications in polydrug-dependent individuals. METHODS: We conducted the first ecological momentary assessment (EMA) study to investigate how smoking is related to other drug use and craving during daily life. For up to 20 weeks, 106 methadone-maintained outpatients carried PalmPilots (PDAs). They reported their craving, mood, behaviors, environment, and cigarette-smoking status in 2 to 5 random-prompt entries/day and initiated PDA entries when they used cocaine or heroin or had a discrete episode of craving for cocaine or heroin. RESULTS: Smoking frequency increased linearly with random-prompt ratings of tobacco craving, cocaine craving, and craving for both cocaine and heroin. Smoking frequency was greater during discrete episodes of cocaine use and craving than during random-prompt reports of low craving for cocaine. This pattern was also significant for dual cocaine and heroin use and craving. Smoking and tobacco craving were each considerably reduced during periods of urine-verified abstinence from cocaine, and there was a (nonsignificant) tendency for morning smoking to be especially reduced during those periods. CONCLUSIONS: This EMA study confirms that smoking and tobacco craving are strongly associated with the use of and craving for cocaine and heroin. Together with prior findings, our data suggest that tobacco and cocaine may each increase craving for (and likelihood of continued use of) themselves and each other. Treatment for tobacco dependence should probably be offered concurrently with (rather than only after) initiation of treatment for other substance-use disorders.

Breath carbon monoxide and semiquantitative saliva cotinine as biomarkers for smoking.

OBJECTIVE: As a biomarker of smoking, semiquantitative analysis of cotinine (NicAlert) offers several advantages over breath carbon monoxide (CO) and quantitative analysis of cotinine. Recent studies have used urine NicAlert and breath CO in combination to verify abstinence. However, no studies have evaluated the performance of saliva NicAlert against or in combination with breath CO. METHOD: Breath CO, saliva NicAlert, and smoking history were compared in an urban population of daily smokers (n = 24) and nonsmokers (n = 25). RESULTS: Saliva NicAlert predicted self-reported smoking with 100% sensitivity and 96% specificity. At a cutoff of > 5 ppm, breath CO had 100% sensitivity and 100% specificity in predicting self-reported smoking. Breath CO was positively correlated with saliva NicAlert and negatively correlated with minutes since last cigarette. CONCLUSION: Saliva NicAlert had high sensitivity and specificity in identifying daily smokers. Compared to saliva NicAlert, breath CO level was more indicative of recent smoking. Future treatment studies should evaluate the performance of saliva NicAlert as an alternative to the urine test.