The radiosynthesis of [11
C]MS-275 was based on our recent report for direct incorporation of 11
into the carbamate carbon (23
). We have previously shown that among common tertiary amines, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was effective at both trapping 11
and C-11 carbamate formation with an amine and an alkyl halide. In the case of MS-275, the required alkyl chloride (3-picolyl chloride, 4
) was commercially available as the hydrochloride salt. Thus, only the amine portion needed to be prepared. Although the precursor amine (3
) could be accessed through hydrogenolysis of MS-275, we obtained the amine (3
) using a straightforward sequence outlined in Figure . A sample of MS-275 was prepared as a reference standard according to the literature (24
Synthesis of the labeling precursor and [11C]MS-275. Reagents and conditions: (i) CDI, THF, reflux, 3 h, 50%; (ii) TFA, CHCl3, rt, 2 h, 96% then HCl, Et2O; (iii) 11CO2, DBU, DMF, 75 °C, 7 min, 25% RCY.
By using the direct fixation labeling method for carbon-11 incorporation, we were able to avoid protection of the aryl amine of precursor 3. While both alkyl amines and aryl amines are competent in the 11CO2 direct incorporation method, alkyl amines are superior substrates, and we noted exclusive formation of the desired product without competition from the aryl amine. In one pot, a solution of precursors 3 and 4 (both as hydrochloride salts in DMF) were combined with excess DBU. After 11CO2 was trapped and the reaction solution was warmed to 75 °C for 7 min, excess 11CO2 was removed and [11C]MS-275 was isolated by reversed-phase chromatography. Given the short half-life of carbon-11 (20.4 min), this one-pot, one-step procedure (without protecting groups) was highly effective providing >20 mCi of [11C]MS-275 per synthesis with relative ease. We expect analogous reaction conditions could be used for the preparation of MS-275 containing other carbon isotopes (13C and 14C), which may prove valuable tools in epigenetic research.
C]MS-275 available, we examined its lipophilicity and plasma protein binding in vitro
. Both the log D
(1.79 ± 0.08, n
= 6) and plasma protein binding (PPB, 7.10% unbound, n
= 2) appeared suitable for BBB penetration (25
). Our data suggested a lower free fraction of MS-275 in plasma than previously reported. However, previous investigations of MS-275 plasma protein binding noted substantial species differences, which may in part explain this difference given our use of the baboon model (26
). Given the literature precedence and our analysis of the physical properties of MS-275, including its molecular weight, lipophilicity, and PPB character, we fully anticipated good BBB penetration in vivo
Using PET, we determined that [11
C]MS-275 exhibited very poor initial BBB penetration and low brain uptake over the scanning time (90 min) when administered intravenously to a baboon, Figure . This result was reproduced in two different animals. A concentration of less than 0.001% of the injected dose per cubic centimeter was distributed in the brain tissue. Coregistration of the PET image with an MRI of the same animal indicated that the areas appearing to have a high level of radioactivity corresponded to the ventricles and are likely outside of the BBB. As an initial probe of the cause of poor BBB uptake, verapamil (0.5 mg/kg iv, 5 min prior to [11
C]MS-275) was used to block P-glycoprotein efflux (verapamil is a P-gp substrate that has been used to increase BBB penetration of other compounds; for recent examples, see refs (27
)). However, we did not observed a change in [11
C]MS-275 BBB permeability and retention nor the overall pharmacokinetics of [11
C]MS-275 with verapamil pretreatment. Although our data suggest that P-gp may not be a major mechanism of MS-275 brain exclusion, further in vitro
and in vivo
experiments will be required to rule out P-gp mediated efflux and to determine what other mechanisms are at play.
[11C]MS-275 PET Imaging (baboon brain): (a) summed PET images (2−90 min) following injection of [11C]MS-275 (4.85 mCi); (b) PET image superimposed with a structural MRI of the brain from the same baboon.
After [11C]MS-275 was administered intravenously, carbon-11 was cleared from circulation by both the renal and biliary systems. Following a 90 min dynamic PET scan of the brain, [11C]MS-275 and its labeled metabolites were primarily found in the urinary bladder (as determined by a segmented rectilinear scan, data not shown). A dynamic scan of the baboon’s torso following [11C]MS-275 administration indicated that in addition to the bladder, radioactivity accumulated in the kidneys, liver, and gallbladder (Figure ).
Time−activity curves derived from PET imaging data for peripheral organs in the baboon torso following administration of [11C]MS-275 (4.74 mCi).
Arterial plasma samples were collected during each of the four PET scans, and the percentage of carbon-11 associated with [11
C]MS-275 was determined using HPLC, Figure . After 40 min, approximately 60% of the radioactivity in plasma was still associated with [11
C]MS-275. Given orally, MS-275 was reported to have good metabolic stability and a long circulation half-life (mean terminal half-life, 33.9 ± 26.2 h) (30
). However, both dose and frequency of administration affect the circulation half-life (52−150 h) (31
). Our study provides rate data for initial clearance at much earlier time points than previous studies and is consistent with rapid initial clearance previously observed. The persistence of unmetabolized MS-275 at later time points (>60 min), as well as the continuing rise of the plasma integral (AUC) over time, is also consistent with the previous pharmacokinetic data reported (30
Baboon plasma analysis: (●) percentage (of total radioactivity) of [11C]MS-275 in plasma over time; (○) metabolite-corrected plasma integral.
Given that [11
C]MS-275 exhibited poor BBB penetration in baboon and that several reports have cited the ability of MS-275 to cross the BBB in rodents (20
), we became interested in determining whether species or dose effects or both existed for MS-275 entry and accumulation in the brain. Using small animal PET imaging, we evaluated the pharmacokinetics of [11
C]MS-275 (adminstered iv) in the rat brain before and after administration of MS-275 (40 mg/kg ip), Figure . As with the baboon, [11
C]MS-275 showed very little BBB penetration and brain accumulation (<0.10% ID/cm3
after only 3 min). The pharmacokinetics of [11
C]MS-275 were not affected by the administration of a large dose of cold (i.e., nonlabeled) MS-275. It is important to note that the clinical dose of MS-275 (6 mg/m2
po) used to treat solid tumors and lymphomas is at least 2 orders of magnitude lower
than the doses used in previous reports of MS-275 brain penetration and CNS effects and in this study.
Figure 6 Rodent imaging experiments with [11C]MS-275. (a) Summed PET images (1−90 min) following injection of [11C]MS-275: (top) baseline scan (1.74 mCi); (bottom) image acquired following pretreatment with MS-275 (40 mg/kg ip; 1.39 mCi). Images are dose (more ...)
To further investigate the effect of MS-275 dose on BBB penetration, we coadministered [11
C]MS-275 and MS-275 to mice subcutaneously. Our choice of dose, route of administration, and tissue sampling time was based on the work by Simonini et al. (20
), which demonstrated that MS-275 altered histone acetylation in several of brain regions. We administered nominally 1.0 mCi of [11
C]MS-275 alone or with either 0.5 mg or 1.0 mg of MS-275 (all animals were 22 ± 1 g). This very high dose corresponds to approximately 0, 25, and 50 mg/kg (0, 60, 120 μmol/kg). We sacrificed each animal 2 h after the injection and determined the amount of radioactivity in blood and brain, Figure . We did not observe a change in the ratio of radioactivity in the brain to blood as a function of dose. The ratio for each dose was ~0.12−0.15, again demonstrating the poor BBB penetration of MS-275. Metabolite analysis of brain homogenate from two animals (one administered [11
C]MS-275 only and one coadministered with 1.0 mg MS-275) indicated that 80% of the radioactivity in the brain was associated with [11
Figure 7 Ratio of radioactivity in brain and blood (at 2 h) for mice coadministered [11C]MS-275 with nonradioactive MS-275 at two doses: indicates injection of [11C]MS-275 only.
Although the brain/blood ratio was consistent from one animal to another, the overall amount of radioactivity that accumulated in the brain varied greatly from animal to animal (even within a group given a particular dose). Using the absolute amount of radioactivity in the brain and the known mass injected, we determined the mass of MS-275 in the brain at 2 h for each given dose. At 60 μmol/kg, there was 326 ± 105 ng of MS-275 per gram of brain tissue at 2 h. At 120 μmol/kg, there was 709 ± 312 ng of MS-275 per gram of brain tissue at 2 h. This calculation assumes that all of the radioactivity in the brain is [11C]MS-275 and is therefore an upper limit measurement of the actual value.
We surmise that although MS-275 BBB penetration is very poor at high doses such as those used by Simonini et al. (20
), sufficient quantities may reside in the brain for the HDAC inhibition observed. However, at the doses of MS-275 used clinically, it is unlikely that a high enough concentration of MS-275 enters the brain to alter histone acetylation (at least acutely). The effect of chronic MS-275 administration at low doses on histone acetylation in the brain will require further investigation.