Topically applied novel TRPV1 receptor antagonist, ACD440 Gel, reduces evoked pain in healthy volunteers, a randomized, double-blind, placebo-controlled, crossover study.

BACKGROUND: The TRPV1 receptor is a key molecule in pain generation. Previous development of oral TRPV1-antagonists was halted due to systemic heat insensitivity and body temperature alterations. The present Phase 1b study investigated the efficacy, safety and plasma exposure of a topically administered TRPV1-antagonist (ACD440 Gel) in healthy subjects. METHODS: The study comprised two parts. In part 1, 24 healthy subjects were included in this randomized double-blind, placebo-controlled, crossover trial. ACD440 Gel or Placebo was applied once daily and wiped off after 1 h, for 5 consecutive days. Assessments were done in normal skin, skin optimized for penetration (by stripping and occlusive gel application) and UVB-irradiated skin. Pain induced by thermo-nociceptive CO2 laser impulses generated laser-evoked potentials (LEPs), with readouts of peak-to-peak (PtP) amplitude in vertex-EEG and pain assessments by VAS (0-100). Endpoints include effects at 1 hour post-dose, AUC(Days 1-5) and AUC(0-24, Day 4). In UVB-irradiated skin, also pain on pinprick and skin redness were assessed. Part 2 explored the plasma pharmacokinetics of ACD440. RESULTS: ACD440 Gel reduced LEP PtP amplitude and VAS pain, p < 0.001, in all skin conditions, versus placebo. In UVB-irradiated skin, pinprick pain was also reduced, p = 0.047. Effects were significant after 1 h, maintaining for at least 9 h. There were no adverse events or drug-induced erythema. Plasma exposures of ACD440 were too low to establish an elimination half-life of ACD400. CONCLUSIONS: Topical ACD440 Gel demonstrated a significant analgesic effect on LEP, VAS score and pinprick pain, with low systemic exposures, supporting further clinical development. SIGNIFICANCE: This study demonstrates that the topical administration of a TRPV1-antagonist, ACD440 Gel, has potential as a new treatment for painful conditions affecting the skin, such as chronic peripheral neuropathic pain, without any local or systemic side effects.

Safety, Tolerability, Pharmacokinetics and Quantitative Electroencephalography Assessment of ACD856, a Novel Positive Allosteric Modulator of Trk-Receptors Following Multiple Doses in Healthy Subjects.

BACKGROUND: ACD856 is a positive allosteric modulator of tropomyosin receptor kinase (Trk) receptors which has shown to have pro-cognitive and anti-depressant-like effects in various animal models. It is currently in clinical development for the treatment of Alzheimer's disease and other disorders where cognition is impaired and is also considered for indications such as depression or other neuropsychiatric diseases. ACD856 has a novel mechanism of action modulating the activity of the Trk-receptors, resulting in increased stimulation of the neurotrophin signaling pathways. Previous studies applying single intravenous and oral doses of ACD856 indicate that ACD856 is safe and well-tolerated by healthy volunteer subjects, and that it has suitable safety and pharmacokinetic properties for further clinical development. OBJECTIVES: To investigate the safety and tolerability of 7 days of treatment with multiple ascending oral doses of ACD856 in healthy subjects, and to characterize its pharmacokinetic (PK) properties. In addition, pharmacodynamic effects of ACD856 using quantitative electroencephalography (qEEG) as an indicator for central target engagement were assessed. DESIGN: This was a prospective, phase I, double-blind, parallel-group, placebo-controlled, randomized study of the safety, tolerability, PK and pharmacodynamics of multiple ascending oral doses of ACD856 in healthy subjects. ACD856 or placebo were administered in 3 ascending dose cohorts of 8 subjects. Within each cohort, subjects were randomized to receive either ACD856 (n=6) or placebo (n=2). SETTING: The study was conducted at a First-in-Human unit in Sweden. PARTICIPANTS: Twenty-four healthy male and female subjects. INTERVENTION: The study medication was administered as an oral solution, with ACD856 or the same contents without the active ingredient (placebo). The dose levels ranged from 10 mg to 90 mg. ACD856 was administered once daily for 7 days, targeting steady state. MEASUREMENTS: Safety and tolerability assessments included adverse events, laboratory, vital signs, 12-lead electrocardiogram (ECG), physical examination, assessment of stool frequency and questionnaires to assess symptoms of anxiety, depression, as well as suicidal ideation and behavior. In addition, cardiodynamic ECGs were extracted to evaluate cardiac safety. PK parameters were calculated based on measured concentrations of ACD856 in plasma, urine, and cerebrospinal fluid (CSF) samples. Metabolite profiling, characterization and analysis was performed based on and urine samples. qEEG was recorded for patients in the two highest dose cohorts (30 and 90 mg/day) as a pharmacodynamic assessment to explore central target engagement. RESULTS: Treatment with ACD856 was well tolerated with no serious adverse events. No treatment emergent or dose related trends were observed for any of the safety assessments. ACD856 was rapidly absorbed and reached maximum plasma exposure at 30 to 45 minutes after administration. Steady state was reached before Day 6, with an elimination half-life at steady state of approximately 20 hours. At steady state, ACD856 exhibited accumulation ratios for Cmax and AUC of approximately 1.6 and 1.9 respectively. The exposure, Cmax and AUC0-24, increased proportionally with the dose. There was no unchanged ACD856 detected in urine. The metabolic pattern in urine and plasma was similar, and in alignment with the metabolites observed in preclinical toxicology studies. The level of ACD856 measured in CSF at steady state increased with dose, indicating Central Nervous System (CNS) exposure at relevant levels for pharmacodynamic effects. ACD856 demonstrated significant dose-dependent treatment-associated changes on qEEG parameters. Specifically, increase of the relative theta power and decrease of the fast alpha and beta power was observed, leading to an acceleration of the delta+theta centroid and an increase in the theta/beta ratio. CONCLUSIONS: ACD856 was well tolerated at the tested dose levels (10-90 mg/daily for 7 days) in healthy subjects. The compound has a robust pharmacokinetic profile, with rapid absorption and dose-dependent exposure. ACD856 was shown to pass the blood-brain-barrier, reach relevant exposure in the CNS and to induce dose-dependent treatment-related changes on qEEG parameters, indicating central target engagement.

Preclinical characterization of three transient receptor potential vanilloid receptor 1 antagonists for early use in human intradermal microdose analgesic studies.

BACKGROUND: The transient receptor potential vanilloid receptor 1 (TRPV1) is a nonselective cation channel involved in the mediation of peripheral pain to the central nervous system. As such, the TRPV1 is an accessible molecular target that lends itself well to the understanding of nociceptive signalling. This study encompasses preclinical investigations of three molecules with the prospect to establish them as suitable analgesic model compounds in human intradermal pain relief studies. METHODS: The inhibitory effectiveness was evaluated by means of in vitro assays, TRPV1 expressing Chinese hamster ovary cells (CHO-K1) and rat dorsal root ganglion cultures in fluorescent imaging plate reader and whole cell patch clamp systems, as well as in vivo by capsaicin-evoked pain-related behavioural response studies in rat. Secondary pharmacology, pharmacokinetics and preclinical safety were also assessed. RESULTS: In vitro, all three compounds were effective at inhibiting capsaicin-activated TRPV1. The concentration producing 50% inhibition (IC50 ) determined was in the range of 3-32 nmol/L and 10-501 nmol/L using CHO-K1 and dorsal root ganglion cultures, respectively. In vivo, all compounds showed dose-dependent reduction in capsaicin-evoked pain-related behavioural responses in rat. None of the three compounds displayed any significant activity on any of the secondary targets tested. The compounds were also shown to be safe from a toxicological, drug metabolism and pharmacokinetic perspective, for usage in microgram doses in the human skin. CONCLUSION: The investigated model compounds displayed ideal compound characteristics as pharmacological and translational tools to address efficacy on the human native TRPV1 target in human skin in situ. SIGNIFICANCE: This work details the pharmaceutical work-up of three TRPV1-active investigational compounds, to obtain regulatory approval, for subsequent use in humans. This fast and cost-effective preclinical development path may impact research beyond the pain management area, as it allows human target engagement information gathering early in drug development.

Clinical testing of three novel transient receptor potential cation channel subfamily V member 1 antagonists in a pharmacodynamic intradermal capsaicin model.

BACKGROUND: The transient receptor potential cation channel subfamily V 1 (TRPV1) is involved in nociception and has thus been of interest for drug developers, as a target for novel analgesics. However, several oral TRPV1 antagonists have failed in development, and novel approaches to target TRPV1 with innovative chemistry are needed. METHOD: This work describes an intradermal microdosing approach in humans for pharmacodynamic deductions and pharmacological profiling of compounds. First, a human capsaicin model was developed, to generate pharmacodynamic translational data (Study Part A, n = 24). Then, three small molecule TRPV1 antagonists (AZ11760788, AZ12048189 and AZ12099548) were investigated in healthy volunteers (Study Part B, n = 36), applying the established model. Pain and flare were assessed by Visual Analogue Score and laser Doppler, respectively. RESULTS: The developed model proved useful for pharmacologic deductions; all compounds caused a dose-dependent inhibition of capsaicin-induced pain and flare responses, with a rank order potency of AZ11760788 > AZ12048189 â‰« AZ12099548. In addition, the dose-response data showed that the minimal antagonist concentrations needed to inhibit TRPV1 was ≥6-7 times the equilibrium dissociation constant for each compound. CONCLUSION: With careful design of a pharmacodynamic translational human pain model, it was possible to rank order TRPV1 efficacy among three investigational TRPV1 antagonists, and to estimate human efficacious concentrations. SIGNIFICANCE: This fast and cost-effective translational approach allows for generation of human target engagement information early in drug development. This could be of value for other development programmes where pharmacological targets are expressed in peripheral sensory nerves.

Identification of 5-S-cysteinyldopa by high performance liquid chromatography in biopsies from patients with dysplastic melanocytic nevi.

The appearance of 5-[(L)-S-cysteinyl]dopa, a major product in pheomelanogenesis was examined in affected and nonaffected skins from 20 patients with clinical signs of dysplastic melanocytic nevi. Analysis by high performance liquid chromatography and electrochemical detection showed that 20 of the 35 lesions had a pathological formation of 5-[(L)-S-cysteinyl]dopa (0.04-28.86 ng/micrograms acid soluble protein). 5-[(L)-S-cysteinyl]dopa was not detected in any of the normal uninvolved skin samples analyzed.

Stimulation of pheomelanogenesis in cultured B16 melanoma cells by 4-tertiary butylcatechol.

Intermediates of pheomelanin in tissue cultured B16 melanoma cells were analyzed by high performance liquid chromatography, and reduced glutathione (GSH), L-dopa, 2-[(L)-S-cysteinyl]-L-dopa (2-SCD) and 5-[(L)-S-cysteinyl]-L-dopa (5-SCD) were quantified. The effects of 4-tertiary butylcatechol (TBC), an antioxidant which causes skin depigmentation, on the levels of the intermediate were then examined. A concentration of 10(-4) M TBC increased the intracellular levels of GSH, 2-SCD and 5-SCD, whereas the L-dopa level was unchanged. The time-course of the increased intermediates corresponded to the elevation of glutathione-metabolizing enzyme activities previously reported by Kawashima et al. [J. invest. Derm. 82, 53 (1984)] in the same cell line exposed to 10(-4) M TBC. The findings establish chemical evidence that TBC stimulates pheomelanogenesis in melanocytes.

Prolonged apparent half-life of delta 1-tetrahydrocannabinol in plasma of chronic marijuana users.

The aim of this study was to characterize the elimination half-life of delta 1-tetrahydrocannabinol in blood plasma in chronic marijuana users. The subjects smoked four cigarettes during a two day period, each cigarette containing 15 mg deuterium-labelled delta 1-tetrahydrocannabinol. The plasma concentrations of deuterium-labelled tetrahydrocannabinol were measured for 13 days using gas chromatography-mass spectrometry equipped with selected ion monitoring. The elimination half-life for delta 1-tetrahydrocannabinol in blood plasma was calculated to be 4.1 +/- 1.1 days (range 2.9-5.0 days) from the two week plasma level curves. Albeit the present results are based upon a small sample, an elimination half-life of delta 1-tetrahydrocannabinol in blood plasma of about 4 days is more in line with apparent half-life excretion of delta 1-tetrahydrocannabinol metabolites in the urine of chronic marijuana smokers.

Urinary excretion half-life of delta 1-tetrahydrocannabinol-7-oic acid in heavy marijuana users after smoking.

The urinary excretion of the total amount of delta 1-tetrahydrocannabinol (delta 1-THC) metabolites, with special emphasis on delta 1-tetrahydrocannabinol-7-oic acid (delta 1-THC-7-oic acid), was studied in thirteen heavy Cannabis users after smoking administration of delta 1-THC, followed by a four week discontinuation period. The total amount of delta 1-THC metabolites and the levels of delta 1-THC-7-oic acid could be followed up to 25 days after abstinence using EMIT d.a.u. cannabinoid assay and high-performance liquid chromatography (HPLC). The urinary excretion half-life, calculated from the concentrations of delta 1-THC-7-oic acid versus time, ranged from 0.8-9.8 days with a mean (+/- SD) of 3.0 +/- 2.3 days. Most of the delta 1-THC-7-oic acid was excreted as conjugate and only trace amounts of unconjugated delta 1-THC-7-oic acid were detected. The total concentrations of delta 1-THC-7-oic acid in urine were compared to the concentrations of "cross-reacting cannabinoids", within the linear range of 20-75 ng/mL, obtained in the semiquantitative EMIT d.a.u. cannabinoid assay. The average ratio of "EMIT concentrations"/delta 1-THC-7-oic acid concentrations obtained by HPLC analysis was 1.23 +/- 84% (C.V.) for 78 urine samples. A total of 83% of the samples with positive EMIT levels (cutoff 20 ng/mL) was confirmed by HPLC analysis (cutoff 7 ng/mL).

Human urinary excretion profile after smoking and oral administration of [14C]delta 1-tetrahydrocannabinol.

The urinary excretion profiles of delta 1-tetrahydrocannabinol (delta 1-THC) metabolites have been evaluated in two chronic and two naive marijuana users after smoking and oral administration of [14C]delta 1-THC. Urine was collected for five days after each administration route and analyzed for total delta 1-THC metabolites by radioactivity determination, for delta 1-THC-7-oic acid by high-performance liquid chromatography, and for cross-reacting cannabinoids by the EMIT d.a.u. cannabinoid assay. The average urinary excretion half-life of 14C-labeled delta 1-THC metabolites was calculated to be 18.2 +/- 4.9 h (+/- SD). The excretion profiles of delta 1-THC-7-oic acid and EMIT readings were similar to the excretion profile of 14C-labeled metabolites in the naive users. However, in the chronic users the excretion profiles of delta 1-THC-7-oic acid and EMIT readings did not resemble the radioactive excretion due to the heavy influence from previous Cannabis use. Between 8-14% of the radioactive dose was recovered in the urine in both user groups after oral administration. Lower urinary recovery was obtained both in the chronic and naive users after smoking--5 and 2%, respectively.

A GC-MS study of three major acidic metabolites of delta 1-tetrahydrocannabinol.

The major urinary metabolite of delta 1-tetrahydrocannabinol (delta 1-THC) (1), delta 1-THC-7-oic acid (2), has been extensively studied for several purposes, including testing in the workplace for drug abuse. Immunoassays in combination with more specific methods such as gas chromatography-mass spectrometry (GC-MS), are commonly used for verification of positive results in the screening. Two additional and recently synthesized acidic metabolites of 1, 4",5"-bisnor-delta 1-THC-7,3"-dioic acid (3) and 4"-hydroxy-delta 1-THC-7-oic acid (4), were studied to widen the scientific basis in the analysis. Five different derivatives were examined using GC-MS. In addition, a new deuterated internal standard for 2, [2H10]-2, was evaluated. According to our results, suitable derivatives of 2, 3, and 4, according to chromatographic properties, are the methyl ester/silyl ether (procedure a), the methyl ester/trifluoroacetate (procedure b), or the silyl ester/silyl ether (procedure c). The estimated recoveries of [2H5]-3 and [2H6]-4 using liquid-liquid extraction were 24% and 50%, respectively. The properties of [2H10]-2 as internal standard were equivalent to those of [2H9]-2 and, under the conditions used, did not appear to give rise to a significantly higher chromatographic resolution from that of 2. However, [2H10]-2 produces ions at different mass numbers, which makes it useful as a complement to the existing deuterated internal standards of 2.

The impact of the site of blood sampling on pharmacokinetic parameters following sublingual dosing to dogs.

INTRODUCTION: Drugs are most commonly administered orally, but some potential drug candidates are not suited for oral administration due to poor absorption, high first pass metabolism or gastrointestinal side effects. The interest for transmucosal dosing for systemic drug delivery is increasing, e.g. buccal, sublingual and nasal routes. The evaluation of the systemic plasma concentration and the derivation of the pharmacokinetic parameters of candidate compounds in preclinical studies are essential for drug development. The effect of site of blood sampling on the measured drug concentration, in both animals and humans, is to some extent known but it is not always taken into consideration in the design of pharmacological and toxicological studies. METHODS: Blood samples were collected both from leg and jugular veins from beagle dogs following a single sublingual dosing of Compound A in order to determine the impact of different sites of blood sampling on plasma pharmacokinetics. Plasma was prepared by centrifugation and plasma concentrations of Compound A were determined by protein precipitation and liquid chromatography followed by mass spectrometric detection. The pharmacokinetic parameters were calculated by non-compartment methods. RESULTS: Sampling from the jugular vein resulted in higher and more variable exposure during the absorption phase compared to sampling from a leg vein. The plasma exposure in the jugular vein, in terms of C(max), was 4-fold compared to that in the leg vein and an approximately 2-fold bioavailability was observed. DISCUSSION: The aim of this investigation was to determine the impact of the different sites of blood sampling on assessing systemic plasma exposure and pharmacokinetic parameters for Compound A following sublingual dosing to dogs. The results demonstrate the significant impact that the site of blood sampling has on PK parameters, and raise concerns of using the jugular vein as a site of sampling after sublingual and other transmucosal routes of dosing in the head region.

A urinary metabolite of delta 1-tetrahydrocannabinol. The first synthesis of 4",5"-bisnor-delta 1-tetrahydrocannabinol-7,3"-dioic acid, and a deuterium labelled analogue.

The first synthesis of unlabelled and [2H5]-labelled 4",5"-bisnor-delta 1-THC-7,3"-dioic acid, the major dicarboxylated urinary metabolite of delta 1-THC in man, is presented (preliminary results of this work have been presented in part at the Melbourne Symposium on Cannabis, Australia, September 1987, Ref. 1). The synthesis of methyl 3-(3,5-dihydroxyphenyl)-[3,3-2H2]-propanoate (8) is described in a nine step sequence from 3,5-dimethoxybenzoic acid in an overall yield of 24%. Compound 8 is condensed with a terpene synthon 9 under acidic conditions, acetylated and hydrolyzed with red HgO and HgCl2 to afford the 1-formyl-4",5",7-trisnor-delta 1-THC-3"-oic acid derivative (11). Compound 11 is oxidized using NaClO2 in 2-methyl-2-butene and hydrolyzed to give (+/-)-4",5"-bisnor-delta 1-THC-7,3"-dioic acid (12). The same approach has been used to prepare both the labelled and unlabelled metabolite.

Glucuronic acid conjugate of delta 1-tetrahydrocannabinol identified in the urine of man.

A delta 1-tetrahydrocannabinol (delta 1-THC) metabolite has been identified as an O-glucuronide of delta 1-THC in the human urine after oral administration of delta 1-THC. The metabolite was present in small amounts and its identity was confirmed by comparison of its mass spectrum to that of a reference compound.

Urinary elimination half-life of delta-1-tetrahydrocannabinol-7-oic acid in heavy marijuana users after smoking.

The urinary excretion of delta 1-tetrahydrocannabinol-7-oic acid (delta 1-THC-7-oic acid), the major urinary metabolite of delta 1-THC, and the total amount of THC metabolites was studied in heavy marijuana users after smoking using high-performance liquid chromatography and the EMT-d.a.u. cannabinoid assay. An average elimination half-life (+/- SD) of 3.0 +/- 2.3 days was obtained for delta 1-THC-7-oic acid. The average ratio (+/- SD) of "EMIT readings"/delta 1-THC-7-oic acid concentrations was 1.23 +/- 1.03.

Lack of metabolic racemisation of ropivacaine, determined by liquid chromatography using a chiral AGP column.

Ropivacaine hydrochloride monohydrate (ropivacaine) is a new local anaesthetic agent which is administered exclusively as the (-)-(S)-form. The aim of the study was to determine whether metabolic racemisation of (-)-(S)-ropivacaine occurs. This was tested in man, rat, dog, and sheep after different routes of administration. The enantiomers of ropivacaine and two of the major metabolites, 3-hydroxy-ropivacaine and 2',6'-pipecoloxylidide (PPX), were determined in urine samples by liquid chromatography on a Chiral AGP column after liquid-liquid extraction. It was possible to detect < 1% of the (+)-(R)-enantiomer of both ropivacaine and the two major metabolites. In the samples examined, no trace of metabolic racemisation was observed. In pharmacokinetic, pharmacodynamic, toxicological, and metabolic studies, therefore, nonchiral assays are considered to be adequate.

Determination of delta 1-tetrahydrocannabinol in human fat biopsies from marihuana users by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric (GC/MS) method for analysis of delta 1-tetrahydrocannabinol (delta 1-THC) in human fat samples is described. The fat sample, obtained from heavy marihuana users 1 week before and 4 weeks after smoking, is homogenized in hexane + 2-propanol, centrifuged, and the supernatant mixed with Lipidex 5000. The solvent is evaporated and the dried gel is packed in a glass column. delta 1-THC is eluted from the column with methanol + water + acetic acid, diluted with water and the eluent is passed through a bed of Octadecylsilane-bonded silica. After washing and drying, the retained delta 1-THC is eluted with hexane, derivatized with N-methyl-N-(t-butyl-dimethysilyl)trifluoroacetamide (MTBSTFA) and finally purified by HPLC on an Octadecyl Sl 100 column in methanol. The amount of delta 1-THC is determined by GC/MS, using selected ion monitoring, and a deuterated internal standard. The recovery of delta 1-THC is about 80%, and the concentration of delta 1-THC in the fat samples analysed ranged between 0.4 and 193 ng/g wet tissue.

Liquid chromatographic bioanalytical determination of ropivacaine, bupivacaine and major metabolites.

Bioanalytical methods for the determination of ropivacaine, bupivacaine and their major metabolites in urine and blood plasma are presented. Ropivacaine is a new local anaesthetic drug mainly used for surgery and for postoperative pain relief. The samples are hydrolysed and cleaned using solid-phase extraction and analysed using ion-pair reversed-phase liquid chromatography with gradient elution. The analytes are detected using UV at 210 nm. The methods are highly selective and the limits of quantification were 1 microM in urine and 0.1 microM in plasma, respectively. The between-day variance was generally below 3% (RSD).

Unexpected adduct ion formation under chemical ionization conditions.

As part of our efforts to characterize the in vivo metabolic fate of the antihypertensive agent alpha-methyldopa, we have examined the urine of alpha-methyldopa-treated rats with the aid of a direct insertion probe chemical ionization mass spectral assay. The mass spectrum of the sample obtained by chromatographic purification followed by treatment with ethanolic hydrochloric acid and pentafluoropropionic anhydride displayed an intense ion at m/z 812, consistent with the beta-ethoxy-N,O,O,O-tetrakispentafluoropropionyl derivative of 6-hydroxy-alpha-methyl-norepinephrine, a potential aromatic hydroxylation product of the known alpha-methyldopa metabolite alpha-methyl-norepinephrine. Comparison of this spectrum with the spectrum obtained with the corresponding synthetic 6-hydroxy-alpha-methylnorepinephrine, however, ruled out this possibility. A more thorough examination of the mass spectral data established that the ion at m/z 812 observed with the metabolic species was due to the formation of an unexpected adduct ion between a known metabolite of alpha-methyldopa and an impurity ion formed from a common constituent of urine. This paper summarizes the characterization of this adduct ion.

Terminal elimination plasma half-life of delta 1-tetrahydrocannabinol (delta 1-THC) in heavy users of marijuana.

The terminal elimination half-life of delta 1-tetrahydrocannabinol (delta 1-THC) was investigated in eight men who were heavy users of marijuana. A stable isotope assay, following smoking deuterium-labeled delta 1-THC, was used to determine plasma concentrations. In two additional users plasma levels were followed after administration of unlabeled delta 1-THC. The subjects were asked to smoke a "loading dose" of 56 mg delta 1-THC during two days and then abstain from all marijuana use for 4 weeks. The pharmacokinetic behavior was consistent with a multicompartment model with a mean plasma elimination half-life of delta 1-THC of 4.3 days when concentrations were followed for 10-15 days after smoking. In the two subjects with detectable plasma levels during 4 weeks, half-lives of 9.6 and 12.6 days was obtained.