Background
Flumazenil (FMZ) is a functionally silent imidazobenzodiazepine which binds to the benzodiazepine binding site of approximately 75% of the brain γ-aminobutyric acid-A receptors (GABA
A
...Rs). Positron Emission Tomography (PET) imaging of the GABAARs with
11
CFMZ has been used to evidence alterations in neuronal density, to assess target engagement of novel pharmacological agents, and to study disorders such as epilepsy and Huntington’s disease. Despite the potential of FMZ PET imaging the short half-life (
t
1/2
) of carbon-11 (20 min) has limited the more widespread clinical use of
11
CFMZ. The fluorine-18 (
18
F) isotopologue with a longer
t
1/2
(110 min) is ideally suited to address this drawback. However, the majority of current radiochemical methods for the synthesis of
18
FFMZ are non-trivial and low yielding. We report a robust, automated protocol that is good manufacturing practice (GMP) compatible, and yields multi-patient doses of
18
FFMZ.
Results
The fully automated synthesis was developed on the Trasis AllinOne (AIO) platform using a single-use cassette.
18
FFMZ was synthesized in a one-step procedure from
18
Ffluoride, via a copper-mediated
18
F-fluorination of a boronate ester precursor. Purification was performed by semi-preparative radio-HPLC and the collected fraction formulated directly into the final product vial. The overall process from start of synthesis to delivery of product is approximately 55 min. Starting with an initial activity of 23.6 ± 5.8 GBq (
n
= 3) activity yields of
18
FFMZ were 8.0 ± 1 GBq (
n
= 3). The synthesis was successfully reproduced at two independent sites, where the product passed quality control release criteria in line with the European Pharmacopoeia standards and ICH Q3D(R1) guidelines to be suitable for human use.
Conclusion
Reported is a fully automated cassette-based synthesis of
18
FFMZ that is Good Manufacturing Practice (GMP) compatible and produces multi-patient doses of
18
FFMZ.
Nalmefene is an opioid antagonist which as a once-a-day tablet formulation has recently been approved for reducing ethanol intake in alcoholic subjects. In order to address the compliance issue in ...this patient population, a number of potential nalmefene prodrugs were synthesized with the aim of providing a formulation that could provide plasma drug concentrations in the region of 0.5–1.0ng/mL for a one-month period when dosed intramuscular to dogs or minipigs. In an initial series of studies, three different lipophilic nalmefene derivatives were evaluated: the palmitate (C16), the octadecyl glutarate diester (C18-C5) and the decyl carbamate (CB10). They were administered intramuscularly to dogs in a sesame oil solution at a dose of 1mg-eq. nalmefene/kg. The decyl carbamate was released relatively quickly from the oil depot and its carbamate bond was too stable to be used as a prodrug. The other two derivatives delivered a fairly constant level of 0.2–0.3ng nalmefene/mL plasma for one month and since there was no significant difference between these two, the less complex palmitate monoester was chosen to demonstrate that dog plasma nalmefene concentrations were dose-dependent at 1, 5 and 20mg-eq. nalmefene/kg. In a second set of experiments, the effect of the chain length of the fatty acid monoester promoieties was examined. The increasingly lipophilic octanoate (C8), decanoate (C10) and dodecanoate (C12) derivatives were evaluated in dogs and in minipigs, at a dose of 5mg-eq. nalmefene/kg and plasma nalmefene concentrations were measured over a four-week period. The pharmacokinetic profiles were very similar in both species with Cmax decreasing and Tmax increasing with increasing fatty acid chain length and the target plasma concentrations (0.5–1.0ng/mL over a month-long period) were achieved with the dodecanoate (C12) prodrug. These data therefore demonstrate that sustained plasma nalmefene concentrations can be achieved in both dog and minipig using nalmefene prodrugs and that the pharmacokinetic profile of nalmefene can be tuned by varying the length of the alkyl group.
Display omitted
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
There is a substantial body of evidence indicating that beta-amyloid peptides (Abeta) are critical factors in the onset and development of Alzheimer's disease (AD). One strategy for combating AD is ...to reduce or eliminate the production of Abeta through inhibition of the gamma-secretase enzyme, which cleaves Abeta from the amyloid precursor protein (APP). We demonstrate here that chronic treatment for 3 months with 3 mg/kg of the potent, orally bioavailable and brain-penetrant gamma-secretase inhibitor N-cis-4-(4-chlorophenyl)-sulfonyl-4-(2,5-difluorophenyl)cyclohexyl-1,1,1-trifluoromethanesulfonamide (MRK-560) attenuates the appearance of amyloid plaques in the Tg2576 mouse. These reductions in plaques were also accompanied by a decrease in the level of reactive gliosis. The morphometric and histological measures agreed with biochemical analysis of Abeta(40) and Abeta(42) in the cortex. Interestingly, the volume of the plaques across treatment groups did not change, indicating that reducing Abeta levels does not significantly alter deposit growth once initiated. Furthermore, we demonstrate that these beneficial effects can be achieved without causing histopathological changes in the ileum, spleen, or thymus as a consequence of blockade of the processing of alternative substrates, such as the Notch family of receptors. This indicates that in vivo a therapeutic window between these substrates seems possible--a key concern in the development of this approach to AD. An understanding of the mechanisms whereby MRK-560 shows differentiation between the APP and Notch proteolytic pathway of gamma-secretase should provide the basis for the next generation of gamma-secretase inhibitors.
TPA023 is a GABA(A) alpha2/alpha3 subtype-selective modulator which in preclinical species has anxiolytic-like activity but does not produce sedative-like properties and is without abuse potential. ...It has good oral bioavailability in rat and dog but not in rhesus monkey (respective oral bioavailability values of 36, 54, and 1%), and in all the three species the half-life after i.v. administration was relatively short (0.6-1.5 h). The plasma concentrations of TPA023 required to produce 50% receptor occupancy were 21-25, 19, and 9 ng/mL in rats, baboons, and humans, respectively. In man, TPA023 has a half-life of around 3-6h when administered as an immediate release formulation, but exposure was more prolonged when it was formulated into a controlled release, gel extrusion module (GEM) tablet. In vivo metabolism was via t-butyl hydroxylation and N-deethylation. A drug-drug interaction study with itraconazole confirmed in vitro metabolic results implicating CYP3A enzymes as the major contributors to in vivo oxidative metabolism. The maximum tolerated doses in healthy, normal volunteers were 2 and 8mg for the immediate-release and GEM formulations, respectively. A post hoc analysis of three separate Phase IIa studies, all of which were halted prematurely, showed that TPA023 reduced scores on the Hamilton Anxiety Scale to a significantly greater extent than placebo. In addition, TPA023 has recently been reported to produce a trend toward improved cognitive performance in a small group of schizophrenia patients. Collectively, these data demonstrate that the alpha2/alpha3-selective partial agonist efficacy of TPA023 translates into a novel pharmacological profile.
Topoisomerase 1 (TOP1) generates transient nicks in the DNA to relieve torsional stress encountered during the cellular processes of transcription, replication, and recombination. At the site of the ...nick there is a covalent linkage of TOP1 with DNA via a tyrosine residue. This reversible TOP1-cleavage complex intermediate can become trapped on DNA by TOP1 poisons such as camptothecin, or by collision with replication or transcription machinery, thereby causing protein-linked DNA single- or double-strand breaks and resulting in cell death. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a key enzyme involved in the repair of TOP1-associated DNA breaks via hydrolysis of 3′-phosphotyrosine bonds. Inhibition of TDP1 is therefore an attractive strategy for targeting cancer cells in conjunction with TOP1 poisons. Existing methods for monitoring the phosphodiesterase activity of TDP1 are generally gel based or of high cost. Here we report a novel, oligonucleotide-based fluorescence assay that is robust, sensitive, and suitable for high-throughput screening of both fragment and small compound libraries for the detection of TDP1 inhibitors. We further validated the assay using whole cell extracts, extending its potential application to determine of TDP1 activity in clinical samples from patients undergoing chemotherapy.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The GABA(A) receptor alpha2/alpha3 subtype-selective compound 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo4,3-bpyridazine (TPA023; also known as ...MK-0777) is a triazolopyridazine that has similar, subnanomolar affinity for the benzodiazepine binding site of alpha1-, alpha2-, alpha3-, and alpha5-containing GABA(A) receptors and has partial agonist efficacy at the alpha2 and alpha3 but not the alpha1 or alpha5 subtypes. The purpose of the present study was to define the relationship between plasma TPA023 concentrations and benzodiazepine binding site occupancy across species measured using various methods. Thus, occupancy was measured using either in vivo (3)Hflumazenil binding or (11)Cflumazenil small-animal positron emission tomography (microPET) in rats, (123)Iiomazenil gamma-scintigraphy in rhesus monkeys, and (11)Cflumazenil PET in baboons and humans. For each study, plasma-occupancy curves were derived, and the plasma concentration of TPA023 required to produce 50% occupancy (EC(50)) was calculated. The EC(50) values for rats, rhesus monkeys, and baboons were all similar and ranged from 19 to 30 ng/ml, although in humans, the EC(50) was slightly lower at 9 ng/ml. In humans, a single 2-mg dose of TPA023 produced in the region of 50 to 60% occupancy in the absence of overt sedative-like effects. Considering that nonselective full agonists are associated with sedation at occupancies of less than 30%, these data emphasize the relatively nonsedating nature of TPA023.
Rationale
Experimental evidence suggests that the differential behavioral effects of benzodiazepines depend on their relative actions at γ-aminobutyric acid type A (GABA
A
) receptors that contain ...either an α1, α2, α3, or α5 subunit.
Objectives
The present study was aimed at understanding the role of α3 subunit-containing GABA
A
(α3GABA
A
) receptors by examining the behavioral pharmacology of TP003 (4,2′-difluoro-5′-8-fluoro-7-(1-hydroxy-1-methylethyl)imidazo1,2-apyridine-3-ylbiphenyl-2-carbonitrile), which shows functional selectivity for α3GABA
A
receptors.
Methods
First, a conflict procedure was used to assess the anxiolytic-like effects of TP003 and a representative clinically available benzodiazepine. TP003 was also administered before daily periods of sucrose pellet availability to evaluate potential hyperphagic effects. In separate experiments, observable behavioral effects were used to assess the motor and sedative effects of TP003.
Results
Administration of TP003 produced robust anti-conflict effects without the rate-decreasing effects that were observed with the representative benzodiazepine. Unlike the reported effects of benzodiazepines, TP003 did not enhance palatable food consumption. However, increases in observable sleep-associated posture were induced by TP003, as were decreases in some species-typical behaviors (vocalization, locomotion, and environment-directed behaviors). When evaluated for its ability to induce a procumbent posture, TP003 failed to produce an effect.
Conclusions
Based on conflict and observation tests in monkeys, our results suggest that TP003 may have anxiolytic properties but lack ataxic, hyperphagic, and pronounced sedative effects characteristic of classical benzodiazepines. TP003 did induce myorelaxant-like effects and had relatively mild sedative effects. Collectively, these results suggest that α3GABA
A
receptors play an important role in the anxiolytic-like and motor effects of benzodiazepine-type drugs.
Full text
Available for:
DOBA, EMUNI, FIS, FSPLJ, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, VSZLJ, ZAGLJ
Rationale
The lack of potent, selective, brain penetrant Y
2
receptor antagonists has hampered in vivo functional studies of this receptor.
Objective
Here, we report the in vitro and in vivo ...characterization of JNJ-31020028 (
N
-(4-{4-2-(diethylamino)-2-oxo-1-phenylethylpiperazin-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide), a novel Y
2
receptor antagonist.
Methods
The affinity of JNJ-31020028 was determined by inhibition of the PYY binding to human Y
2
receptors in KAN-Ts cells and rat Y
2
receptors in rat hippocampus. The functional activity was determined by inhibition of PYY-stimulated calcium responses in KAN-Ts cells expressing a chimeric G protein Gqi5 and in the rat vas deferens (a prototypical Y
2
bioassay). Ex vivo receptor occupancy was revealed by receptor autoradiography. JNJ-31020028 was tested in vivo with microdialysis, in anxiety models, and on corticosterone release.
Results
JNJ-31020028 bound with high affinity (pIC
50
= 8.07 ± 0.05, human, and pIC
50
= 8.22 ± 0.06, rat) and was >100-fold selective versus human Y
1
, Y
4
, and Y
5
receptors. JNJ-31020028 was demonstrated to be an antagonist (pK
B
= 8.04 ± 0.13) in functional assays. JNJ-31020028 occupied Y
2
receptor binding sites (~90% at 10 mg/kg) after subcutaneous administration in rats. JNJ-31020028 increased norepinephrine release in the hypothalamus, consistent with the colocalization of norepinephrine and neuropeptide Y. In a variety of anxiety models, JNJ-31020028 was found to be ineffective, although it did block stress-induced elevations in plasma corticosterone, without altering basal levels, and normalized food intake in stressed animals without affecting basal food intake.
Conclusion
These results suggest that Y
2
receptors may not be critical for acute behaviors in rodents but may serve modulatory roles that can only be elucidated under specific situational conditions.
Full text
Available for:
DOBA, EMUNI, FIS, FSPLJ, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, VSZLJ, ZAGLJ
In pursuit of a GABAA α5-subtype-selective inverse agonist to enhance cognition, a series of 6,7-dihydro-2-benzothiophen-4(5H)-ones has been identified as a novel class of GABAA receptor ligands. ...These thiophenes have higher binding affinity for the GABAA α5 receptor subtype compared to the GABAA α1, α2, and α3 subtypes, and several analogues exhibit high GABAA α5 receptor inverse agonism. 6,6-Dimethyl-3-(2-hydroxyethyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one (43) has been identified as a full inverse agonist at the GABAA α5 receptor and is functionally selective over the other major GABAA receptor subtypes. 43 readily penetrates into the CNS to give selective occupancy of GABAA α5 receptors. In addition, 43 enhances cognitive performance in rats in the delayed ‘matching-to-place' Morris water maze testa hippocampal-dependent memory taskwithout the convulsant or proconvulsant activity associated with nonselective, GABAA receptor inverse agonists.
Studies with our screening lead 5 and the literature compound 6 led to the identification of 6-benzyloxy-3-(4-methoxy)phenyl-1,2,4-triazolo3,4-aphthalazine 8 as a ligand with binding selectivity for ...the γ-aminobutyric acid-A (GABA-A) α3- and α5-containing receptor subtypes over the GABA-A α1 subtype (K i: α2 = 850 nM, α3 = 170 nM, α5 = 72 nM, α1 = 1400 nM). Early optimization studies identified the close analogue 10 (K i: α2 = 16 nM, α3 = 41 nM, α5 = 38 nM, α1 = 280 nM) as a suitable lead for further study. High-affinity ligands were identified by replacing the 6-benzyloxy group of compound 10 with 2-pyridylmethoxy (compound 29), but binding selectivity was not enhanced (K i: α2 = 1.7 nM, α3 = 0.71 nM, α5 = 0.33 nM, α1 = 2.7 nM). Furthermore, on evaluation in xenopus oocytes, 29 was discovered to be a weak to moderate inverse agonist at all four receptor subtypes (α1, −7%; α2, −5%; α3, −16%; α5, −5%). Replacement of the 3-phenyl group of 29 with alternatives led to reduced affinity, and smaller 3-substituents led to reduced efficacy. Methyl substitution of the benzo-fused ring of 29 at the 7-, 8-, and 10-positions resulted in increased efficacy although selectivity was abolished. Increased efficacy and retention of selectivity for α3 over α1 was achieved with the 7,8,9,10-tetrahydro-(7,10-ethano)-phthalazine 62. Compound 62 is currently one of the most binding selective GABA-A α3-benzodiazepine-site partial agonists known, and although its selectivity is limited, its good pharmacokinetic profile in the rat (33% oral bioavailability after a 3 mg/kg dose, reaching a peak plasma concentration of 179 ng/mL; half-life of 1 h) made it a useful pharmacological tool to explore the effect of a GABA-A α2/α3 agonist in vivo.
PDF
