Peter N. Dorff

Absence of direct effects on the dopamine D2 receptor by mGluR2/3-selective receptor agonists LY 354,740 and LY 379,268.

We previously reported the absence of high‐affinity binding of the group II metabotropic glutamate receptor agonists LY 354,740 and LY 379,268 to the D2L dopamine receptor. A rebuttal to our findings has since been reported (see Introduction section); this study represents our response. Analysis by LCMS of LY 354,740 and LY 379,268 used in this study revealed the correct molecular mass for these compounds. Both LY 354,740 and LY 379,268 exhibited potent agonist activity for mGluR2 in the 35S‐GTPγS assay. Functionally, neither compound displayed antagonist activity in the GTPγS assay with recombinant D2. At concentrations up to 10 μM, both compounds failed to displace [3H]‐raclopride, [3H]‐PHNO, or [3H]‐domperidone in filter‐binding assays under isotonic (120 mM NaCl or N‐methyl glucamine) or low‐ionic strength (no NaCl or N‐methyl glucamine) conditions. Some displacement of [3H]‐domperidone (20–40%) was observed at 30 μM of LY 354,740 under low‐ionic strength and under isotonic conditions in the absence of NaCl. No displacement of [3H]‐domperidone was detected in a two site model at lower (<100 nM) concentrations of either compound. Moreover, no D2 activity was observed for LY 354,740 or LY 379,268 in the CellKey™ (cellular dielectric spectroscopy) assay. In this communication, we discuss the possible reasons for differences in our study and the previously published work and implications of these studies for mechanisms of antipsychotic action. Synapse 65:64–68, 2011.

D2 receptor occupancy in conscious rat brain is not significantly distinguished with [3H]‐MNPA, [3H]‐(+)‐PHNO, and [3H]‐raclopride

Positron emission tomography (PET) antagonist ligands such as [11C]‐raclopride are commonly used to study dopamine D2 receptor (D2) binding of antipsychotics. It has been suggested that agonist radioligands bind preferentially to the high‐affinity state of D2 receptor and may provide a more relevant means of assessing D2 occupancy. The main objective of this study was to determine if D2 receptor occupancy (RO) could be differentiated with agonist and antagonist radioligands in vivo. Agonist radioligands [3H]‐MNPA and [3H]‐(+)‐PHNO were synthesized and compared to antagonist [3H]‐raclopride in the in vitro binding and in vivo occupancy studies. In vivo, unanesthetized rats were pretreated with quinpirole (full agonist), aripiprazole (partial agonist), or haloperidol (antagonist) prior to administration of the agonist or antagonist radioligand. All three pretreatment compounds showed equivalent dose‐dependent D2 receptor occupancy in the rat striatum with each radioligand. The in vivo receptor occupancy results suggested that the binding of quinpirole, aripiprazole, and haloperidol to the high or low affinity state of the D2 receptor could not be differentiated using radiolabeled agonists or antagonists, presumably due to a predominance of high affinity states of the D2 receptor in vivo. This hypothesis was supported in part by the in vitro binding results. Our in vitro results show that [3H]‐MNPA binds to D2S transfected CHO cell membranes at a single high affinity site. Displacement of [3H]‐(+)‐PHNO binding by quinpirole and elimination of most [3H]‐(+)‐PHNO binding by the guanine nucleotide GppNHp in striatal membranes suggest that the majority of D2 in striatal tissue is G‐protein coupled. Together, these findings suggest that D2 agonist radioligands produce in vivo receptor occupancy comparable to [3H]‐raclopride. Synapse, 2010.

In vitro assessment of metabolic drug–drug interaction potential of AZD2624, neurokinin-3 receptor antagonist, through cytochrome P450 enzyme identification, inhibition, and induction studies

AZD2624 was pharmacologically characterized as a NK3 receptor antagonist intended for treatment of schizophrenia. The metabolic drug–drug interaction potential of AZD2624 was evaluated in in vitro studies. CYP3A4 and CYP3A5 appeared to be the primary enzymes mediating the formation of pharmacologically active ketone metabolite (M1), whereas CYP3A4, CYP3A5, and CYP2C9 appeared to be the enzymes responsible for the formation of the hydroxylated metabolite (M2). The apparent Km values were 1.5 and 6.3 µM for the formation of M1 and M2 in human liver microsomes, respectively. AZD2624 exhibited an inhibitory effect on microsomal CYP3A4/5 activities with apparent IC50 values of 7.1 and 19.8 µM for midazolam and testosterone assays, respectively. No time-dependent inactivation of CYP3A4/5 activity (midazolam 1′-hydroxylation) by AZD2624 was observed. AZD2624 demonstrated weak to no inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP2D6. AZD2624 was not an inducer of CYP1A2 or CYP2B6. Although AZD2624-induced CYP3A4 activity in hepatocytes, the potential of AZD2624 to cause inductive drug interactions of this enzyme was low at relevant exposure concentration. Together with targeted low efficacious concentration, the results of this study demonstrated AZD2624 has a relatively low metabolic drug–drug interaction potential towards co-administered drugs. However, metabolism of AZD2624 might be inhibited when co-administrated with potent CYP3A4/5 inhibitors.

Metabolism of a G Protein-Coupled Receptor Modulator, Including Two Major 1,2,4-Oxadiazole Ring-Opened Metabolites and a Rearranged Cysteine-Piperazine Adduct

Metabolites of a G protein-coupled receptor modulator containing 1,2,4-oxadiazole and piperazine substructures were identified in vitro in human, rat, and dog hepatocyte incubates and in vivo in rat plasma, bile, urine, and feces by using 14C-radiolabeled parent compound. Exposure coverage for the major circulating metabolites in humans at steady state and in preclinical species used in drug safety assessments was determined by using pooled plasma samples collected from a human multiple ascending dose study and a 3-month rat toxicokinetic study. Metabolites M1 and M2, which were formed by opening of the 1,2,4-oxadiazole ring, were observed as major metabolites both in vitro and in vivo across species. The carboxylic acid metabolite M2 was presumably formed through reductive N-O bond cleavage of the oxadiazole ring and subsequent hydrolysis. However, the mechanism for the formation of the unusual N-cyanoamide metabolite M1 remains uncertain. Neither M1 nor M2 had any target activity, as did parent drug P. In rat bile, rearranged Cys-piperazine and Gly-Cys-piperazine adducts, involving the formation of a five-membered heteroaromatic imidazole derivative from a six-membered piperazine ring, were observed as minor metabolites. These findings support a previously reported mechanism regarding glutathione detoxification for piperazine bioactivation products.