In order to facilitate the investigation of C2 derivatives of salvinorin A using PET, we developed and optimized a procedure to label the C18 methyl ester with carbon-11. O-Demethyl salvinorins A and B have been prepared previously by nucleophilic cleavage with LiI or LiSEt. Although these conditions were not effective when applied to the ethoxymethyl ether, sufficient quantities of the O-demethyl compound were obtained for radiolabeling and imaging studies by ethoxymethylation of O-demethylsalvinorin B.
We found that methylation of the C18 acid with [11C]-methyl iodide could be accomplished in DMSO with only a small amount of Cs2CO3 (heterogeneous). The use of a poorly soluble base minimized the known epimerization of the C8 carbon and led to suitable radiochemical yields for each of the three compounds we pursued. We anticipate this simple procedure will be adaptable to labeling other C2 salvinorin derivatives either with carbon-11, long-lived isotopes (e.g. 3H, 14C) or stable isotopes (e.g. 2H, 13C).
The lipophilicity and plasma protein binding characteristics of each labeled compound were determined, . From these data, it is evident that the C2 position of SA is important in plasma protein binding. A higher proportion of [11C]-SB remained unbound in plasma (more than double that of SA and EOM-SB). The percentage of [11C]-EOM-SB in the free fraction, bound to plasma protein, was higher than was observed for [11C-SA]. Due to this, we would expect at equal plasma concentrations that EOM-SB would be more potent than SA. Thus as metabolism and excretion occur, the increased free fraction also may contribute to an increased duration of action given that EOM-SB will remain above a threshold longer than SA.
Summary of in vitro data (mean ± stdev)
Using a whole brain region of interest, we determined that all three labeled compounds rapidly entered and exited the brain, . Consistent with our data from [acetyl
C]-SA, the maximum average concentration of SA in the brain following bolus intravenous administration was 0.015% ID/cm3
. In the previous study, where metabolism gave 1-[11
C]-acetate (Hooker, 2008
), we observed a small amount of residual radioactivity as late as 60 min after injection (0.004% ID/cm3
), whereas [11
C]-SA (labeled at the methyl ester) cleared to 0.001% by the same time. The major labeled metabolite from [11
C]-SA was [11
C]-SB and thus total brain radioactivity likely represents the sum of the two species. To evaluate this, we labeled SB itself.
Figure 2 Average of whole brain time-activity curves for [11C]-labeled compounds. [11C]-SA (n = 3); ○ [11C]-SB (n = 3); ▲ [11C]-EOM-SB (n =2). (inset) mean+stdev over first 10 min post injection.
[11C]-SB entered and cleared the brain with kinetics similar to [11C]-SA with an approximately 20% lower average maximum concentration (peaking at 70 s and clearing to half of its maximum concentration in 6.0 min). As with [11C]-SA, no accumulation of radioactivity was observed in any brain region over time. [11C]-SB is a potential metabolite of [11C]-EOM-SB, but these data suggested it would not alter the results if formed.
Given the increased affinity and potency of EOM-SB over SA, we expected to see increased specific binding of [11C]-EOM-SB at KORs. However, we found that [11C]-EOM-SB kinetics were similar to [11C]-SA. In fact, [11C]-EOM-SB had a lower average concentration at 30 s after injection than either [11C]-SA or [11C]-SB.
The major difference between [11C]-EOM-SB and [11C]-SA was in the rate of metabolism, . The protecting group imparted increased stability: ~50% of the radioactivity was parent compound at 30 min after injection. This is in stark contrast to [11C]-SA, which is metabolized to ~50% in under 5 min. The integral of metabolite-corrected plasma radioactivity over time for [11C]-EOM-SB continues to climb longer than that of [11C]-SA (), reflecting longer persistence in plasma. The metabolites of [11C]-EOM-SB observed by HPLC were more polar than the parent compound. Co-injection of [11C]-EOM-SB plasma samples with SB on HPLC indicated that some [11C]-SB may be formed, however the percentage of [11C]-SB could not be accurately determined due to the presence of co-eluting [11C]-metabolites. We were not able to exclude the possibility that some of the parent compound had epimerized at the C8 position over time for either [11C]-SB or [11C]-EOM-SB, however incubation of [11C]-EOM-SB in plasma did not cause epimerization. Our data indicate that none of these compounds are suitable candidates for PET imaging of KORs. Due to their low uptake and rapid clearance, PET signal intensity largely reflects plasma drug levels rather than the much lower specific labeling of KORs.
Figure 3 Baboon plasma analysis. During PET scans, samples of plasma were analyzed to determine the percentage of radioactivity associated with the injected compound. [acetyl-11C]-SA (n = 10); ○ [11C]-SB (n = 3); ▲ [11C]-EOM-SB (n = 2). (more ...)
Figure 4 Metabolite corrected plasma integrals. [acetyl-11C]-SA (n = 10); ○ [11C]-SB (n = 3); ▲ [11C]-EOM-SB (n = 2). (inset) error bars for [11C]-SB are shown separately to avoid confusing overlap with [11C]-EOM-SB error bars.
Although our results confirm that SB and EOM-SB are metabolized much more slowly than SA, a similarly dramatic increase in brain residence time was not observed (). Thus, the brief duration of action of SA is not due solely to metabolism, since a less rapidly metabolized derivative and indeed a metabolite itself are removed from brain almost as rapidly. All three of these compounds show low brain uptake, and are rapidly eliminated, which may suggest transport is mediated by transporters at the blood brain barrier. Consistent with this, efflux of salvinorin A by permeability glycoprotein (P-gp) has recently been reported (Teksin et al., 2009
These results were surprising, given the increased duration of analgesia reported for the methoxymethyl ether. This may be due to the different routes of administration. After intravenous administration, as used here, the rate of elimination from the brain and plasma was rapid and thus could mask an effect related to metabolism. Intraperitoneal (i.p.) administration, as used in the analgesia assays (Wang et al., 2008
), introduces a delay, as the drug is gradually released from the injection site. If the drug is not metabolized during the absorption process, the slower route of administration will prolong the drug's effects. Thus, differences in metabolic rate will be more apparent by intraperitoneal administration. To evaluate this, we administered [11
C]-labeled SA and EOM-SB to rats i.p.
The absorption kinetics of the two compounds were nearly identical (as determined by a 2 mm spherical ROI placed in the injection site) as were the kinetics in the rodent brain, . These data were consistent with the brain kinetics obtained using LC-MS for SA administered i.p. (Teksin et al., 2009
). The curves in represent the total radioactivity of all labeled compounds in the brain. To determine the amount of remaining parent compound, we analyzed brain homogenate at ~65 min post injection. The proportion of [11
C]-EOM-SB remaining (14%) was nearly 3-fold higher than for [11
C]-SA. This suggests that compounds with slower metabolism would indeed display longer duration of action by this route.
Figure 5 (a) Average of whole brain time-activity curves for [11C]-labeled compounds administered i.p. to Sprague-Dawley rats. [11C]-SA (n = 2) ▲ [11C]-EOM-SB (n =2). mean±stdev (b) Brain homogenate analysis for parent compound at 60 (more ...)