Salvinorin A was labeled with carbon-11, a positron emitting isotope, and studied using PET, a powerful noninvasive technique to image drug pharmacokinetics in the living brain at concentrations far below pharmacologically active doses (Fowler and Wolf, 1997
). Salvinorin A was isolated from dried S. divinorum
by a procedure similar to those previously described (Munro et. al., 2003
). Precursors for radiolabeling were synthesized by known chemical degradation pathways. Two positions for carbon-11 incorporation were considered: the acetate at C(2) and the methyl-ester at C(18). The methyl ester was an obvious candidate for labeling with [11
C]-methyl iodide, but the precursor synthesis and subsequent methylation was plagued by epimerization (Beguin et. al., 2003). Conversely, the acetate at the C(2) position was a superior labeling site candidate. The C(2) acetate is the most labile functional group of salvinorin A and is believed to be the primary site of metabolic degradation and inactivation (Schmidt, M.S. et. al., 2005
). This is advantageous in imaging studies where metabolic cleavage at the C(2) position would not result in any radioactive diterpene metabolites.
Indeed, the C(2) acetate was removed cleanly using sodium bicarbonate in methanol and afforded salvinorin B (1
) in good purity without chromatography as previously reported (Tidgewell, et. al., 2004
). However, it was later determined that trace impurities left from this process limited radiochemical yields. Consequently, these impurities were removed by iterative chromatography and crystallization for precursor batches used for radiolabeling.
Carbon-11 radiolabeling began with the production [11
C]-acetyl chloride from [11
C]-carbon dioxide using phthaloyl dichloride, a method adapted from the literature (Le Bars et. al., 1987
). The [11
C]-acetyl chloride was carried in a slow stream of argon through a solution of 1
-dimethylaminopyridine in DMF at 0 °C, . The total reaction and purification time from end of bombardment averaged 40 min and radiochemical yields (based on 11
) were 3.5 – 10 % decay corrected to the end of bombardment. Radiochemically, [11
C]-Salvinorin A (2
) was greater than 98 % pure for all imaging experiments and had a specific activity of 0.20 – 0.75 Ci/μmol. The low specific activity was most likely the result of atmospheric CO2
introduced into the Grignard solution during storage and liquid transfers, but could not be improved by fresh preparation and immediate use. Even at this specific activity, the maximum injected dose of salvinorin A was less than 0.35 μg/kg, an order of magnitude lower than measured psychoactive doses of 3.0-7.5 μg/kg when smoked (Siebert, 1994
characterization suggested that [11
C]-salvinorin was suitable for BBB penetration consistent with its reported potency (Siebert, 1994
). The log D of [11
C]-Salvinorin A at pH = 7.4 was 2.3 and 16 % was found in the free fraction in plasma protein binding assays, .
To examine BBB penetration, kinetics and brain distribution, six adult female baboons were administered 1 - 4 mCi of 2
, . Salvinorin A is most commonly abused by smoking fortified leaves or extracts of S. divinorum
(Gonzalez et. al., 2006
; Babu et. al., 2008
). In this way, a large dose can reach the brain within seconds to initiate visual hallucinations that reach full effect in about 30 s (Siebert, 1994
). Intravenous administration of 2
served as a convenient model for the rapid delivery of a smoked drug to the brain. (Intravenous administration has been validated in numerous studies as a substitute for smoking. See for example studies with nicotine by Lux et. al., 1987
). Animals anesthetized with ketamine and maintained with isoflurane were positioned in a PET scanner and given 2
formulated in water with 10% ethanol in paired studies 2 h apart to measure reproducibility of repeated measures and the effects of naloxone. No physiological effects (i.e. changes in blood pressure, heart rate, pO2
, and body temperature) were observed during any of these studies. Dynamic data were collected for 60 min after injection of 2
. For the second scan, a repeat injection was given. In four studies, animals were treated with 1.0 mg/kg naloxone in saline, 15 min prior, to assess binding specificity. Naloxone is a potent antagonist at μ-opioid receptors with lower affinity at κ- and δ-opioid receptors. Although nonspecific, it has been used to block behavioral effects of κ-opioid drugs (Tang and Collins, 1985
) and binding of κ-opioid radiotracers in primates (see for example Talbot, et. al., 2005
Summary of baboon PET studies with [11C]-Salvinorin A
Using a whole brain region of interest, we determined that 2
rapidly entered the brain attaining a maximum concentration in approximately 40 s, . This is significant given that the rate of a drug reaching its peak concentration in the brain has been linked directly to its reinforcing properties and is often as important as the dose (Balster and Schuster, 1973
). For perspective, the input rate we observed was nearly an order of magnitude faster than the input of [11
C]-cocaine, which peaks 5-6 min after intravenous administration (Volkow et. al., 1987). Clearance of C-11 from the brain was also astonishingly rapid with a half-life from peak of 8 min, reaching 25% of maximum in less than 30 min. The kinetics from our studies were consistent with the duration of action of salvinorin A, which is typically less than 10 min (Baggott et. al., 2004
and Siebert, 1994
Fig 2 [11C]-Salvinorin A in Papio anubis baboon brain. Time-activity curves for whole-brain VOIs, baseline () n = 6, mean+sem; pretreated with 1 mg/kg naloxone (□), n = 4, mean+sem. Representative summed frames for individual studies that are (more ...)
The average maximum brain concentration of 2 was 0.0175 %ID/cc which corresponds to 3.3 % of the administered dose in the whole brain. Considering that smoked doses of 200 μg are effective in humans, we can estimate that less than 10 μg in the human brain account for the drug's psychoactive effects, further underscoring the extraordinary potency of salvinorin A. In paired pretreatment studies with naloxone, the average concentration of 2 in the brain was not significantly lower than the baseline scan. Test-retest variability examined in one study was determined to be 15%, with substantial inter-subject variability in the absolute brain concentration (standard error in ).
C]-Salvinorin A was widely distributed throughout the brain in both cortical and subcortical regions, ( and ). The most persistent 11
C-concentration was seen in the spine and throughout the nasal and salivary tracts, . Intriguingly, the highest concentration in the brain in all studies was in the cerebellum. This is particularly interesting given the behavioral effects of salvinorin A when abused and the role of the cerebellum in the integration of sensory perception and motor control. While κ-opioid receptors are distributed throughout the brain, including the cerebellum, the concentration of 2
did not correlate with κ-opioid density determined in previous studies (Talbot et. al., 2005
and references therein). This may indicate a high degree of non-specific binding or could indicate that other specific binding modes exist. Our studies with naloxone, a non-specific opioid antagonist, do not rule out either. Given its visual hallucinogenic properties, it is worth noting that there was also a significant amount of activity in the striate (visual) cortex.
Whole head PET images of [11C]-Salvinorin A from a baseline and naloxone pretreated paired study (BEJ186) from 3-7 min (left) and 20-40 min (right). Images are normalized to injected dose (scale bar is %ID).
Regional Distribution of [11C]-Salvinorin A in baboon braina
Naloxone pretreatment (1 mg/kg i.v.) did not significantly change the concentration of [11C]-salvinorin A in the brain nor did it affect the magnitude of the area under the plasma time-activity curve (i.e. input). In order to examine this phenomenon more thoroughly and to determine if regional variations existed over time, we modeled the kinetic PET data using a variety of methods. We determined that due to the initial fast kinetics but persistent residual activity, a two-compartment model was most appropriate; we used a metabolite corrected plasma input to model time-activity curves. Modeling data were overwhelmed at late time points with noise from low counting statistics resulting in inconsistent and uninterpretable distribution volumes (hence data not shown). At later time points (i.e. 20 – 40 min), the small amount of C-11 remaining (less than 0.005% ID/cc) was still concentrated to the cerebellum. There was very little C-11 observed in white matter regions.
The absence of an effect by naloxone may indicate that [11
C]-salvinorin A distribution was dominated by non-specific binding, that naloxone is not a sufficiently competitive binder at the κ-opioid agonist site, and/or that our specific activity was too low to observe molecular interactions. Taking conservative values for the Bmax
(10 pmol/g), mass injected (10 μg), and dissociation constant (2.5 nM), we calculate that 47% occupancy is represented at our highest activity concentration in the brain i.e. 0.03 %ID/cc (Hume et. al., 1998
). This occupancy, which we the feel is the absolute maximum for our experiments, was well above the 1-5% suggested for suitable tracers (Huang and Phelps, 1986
). However, this level of occupancy does not change our interpretation of the PET imaging data, which we have used to probe salvinorin A pharmacokinetics. To evaluate [11
C]-salvinorin A as a tracer for κ-opioid receptors, more studies would be required.
Blood samples collected during PET studies were used to determine the C-11 concentration in the plasma throughout the 60 min scan. The amount of unmetabolized 2
was determined assuming that radioactive metabolites would be more polar than salvinorin A (i.e. that the predominant metabolism would cleave the C(2) acetate), . Indeed, solid phase extraction of plasma samples resulted in one polar and one non-polar fraction, each of which may represent multiple species. Control experiments demonstrated that [11
C]-salvinorin A eluted in the non-polar fraction. The percent of C-11 in the non-polar fraction from the plasma (n = 7) is shown in . The non-polar fraction of C-11 rapidly decreased to 40% by 5 min. Although we were not able to conclusively identify the number and identity of metabolic species by HPLC, the C-11 clears quickly from the blood indicating that transport to the brain is dominated by the first minute. Moreover, we must note that our kinetic analysis assumes that labeled metabolites had a small contribution to C-11 in the brain within the first ten minutes of imaging. The metabolism rate of 2
did not vary between individuals, nor was there significant variation introduced by naloxone pretreatment (n = 3). In all experiments, radioactivity in the plasma was below 0.01 %ID/cc within 10 min and an order of magnitude lower by 60 min. Our C-11 plasma data were highly consistent with the plasma pharmacokinetics of unlabeled salvinorin A determined in rhesus monkeys (Schmidt, M.D. et. al., 2005
Fig 4 Blood data. (left) Arterial blood was collected from the popliteal artery in 5 s intervals post injection and the activity in the plasma was counted. (right) Fraction of total radioactivity in the plasma attributed to 2 from 7 primates (mean ± (more ...)
A further examination of the pharmacokinetics and distribution 2 and its labeled metabolites in peripheral organs was accomplished using PET, . Initial uptake (concentration) was highest in the kidneys, however due to its size the total amount of C-11 was greatest in the liver. The liver uptake achieved 0.04 %ID/cc min and decreased gradually. Carbon-11 began accumulating in the gallbladder after 10 min, which at 60 min contained 0.12 %ID/cc, the highest of any organ. Our kinetic analysis suggests that at least two modes of metabolism and excretion occur; one through renal filtration (presumably hydrophilic metabolites) and the other through collection in the gall bladder (presumably lipophilic metabolites). Whole body images taken from 65 – 110 min indicate that approximately equal amounts of C-11 were in the gallbladder (and biliary tract) and urinary bladder, . Carbon-11 in the spine endured well beyond the time in the brain and was easily observed two hours after injection. Naloxone did not appear to change the overall distribution of C-11 (based on images 65 – 110 min post injection), however kinetic differences, if any, were not determined.
Time-activity curves for peripheral organs in the baboon torso (BEJ364).