Henrik Pedersen

Identification of the Cytochrome P450 and Other Enzymes Involved in the In Vitro Oxidative Metabolism of a Novel Antidepressant, Lu AA21004

1-[2-(2,4-Dimethyl-phenylsulfanyl)-phenyl]-piperazine (Lu AA21004) is a novel antidepressant that is currently in late-stage clinical development for major depressive disorder. In the present study, the metabolism of Lu AA21004 was investigated using human liver microsomes (HLM), human liver S9 fraction, and recombinant enzymes. Lu AA21004 was found in vitro to be oxidized to a 4-hydroxy-phenyl metabolite, a sulfoxide, an N-hydroxylated piperazine, and a benzylic alcohol, which was further oxidized to the corresponding benzoic acid [3-methyl-4-(2-piperazin-1-yl-phenysulfanyl)-benzoic acid (Lu AA34443)]. The formation of the 4-hydroxy-phenyl metabolite was catalyzed by CYP2D6 with some contribution from CYP2C9, whereas the formation of the sulfoxide was mediated by CYP3A4/5 and CYP2A6. CYP2C9 and CYP2C19 were the primary enzymes responsible for formation of the N-hydroxylated metabolite. The benzylic alcohol was formed by CYP2D6 only. The oxidation of the benzylic alcohol to the corresponding benzoic acid of Lu AA21004 was catalyzed by alcohol dehydrogenase and aldehyde dehydrogenase, with some contribution from aldehyde oxidase. CYP2D6 was also capable of catalyzing the formation of the benzoic acid of Lu AA21004; however, its overall contribution to this pathway was negligible. Enzyme kinetic parameters revealed that the rate-limiting step in the formation of the benzoic acid from Lu AA21004 is the formation of the corresponding alcohol. Thus, the intrinsic clearance (Vmax/Km) in HLM for metabolism of Lu AA21004 to the benzylic alcohol was 1.13 × 10−6 l · min−1 · mg−1, whereas the subsequent metabolism of the benzylic alcohol to the benzoic acid of Lu AA21004 is characterized by an intrinsic clearance (Vmax/Km) in S9 fraction of 922 × 10−6 l · min−1 · mg−1.

Biosynthesis and Identification of an N-oxide/N-glucuronide Metabolite and First Synthesis of an N-O-glucuronide Metabolite of Lu AA21004

This article describes the biosynthesis and identification of a new class of metabolites, a piperazine N-oxide/N-glucuronide metabolite 4-[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]-1-β-d-glucuronic acid-piperazine 1-oxide (4). The metabolite was found in urine and plasma from humans and animals dosed with 1-[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]-piperazine hydrobromide (Lu AA21004, 1), as a novel multimodal antidepressant under development for treatment of depression. Human liver microsomes in combination with uridine 5′-diphosphoglucuronic acid were used as an in vitro system to generate enough material of 4 to perform one- and two-dimensional 1H and 13C NMR experiments for structure elucidation. Based on rotating frame Overhauser enhancement spectroscopy NMR experiments, the distance correlation between a piperazine proton and the anomeric proton of the glucuronic acid moiety is of a magnitude similar to that of the H-3′ and H-5′ protons and can only be explained by proximity in space and the postulated structure (4). The structural analog, the N-O-glucuronic acid conjugate 6-{4-[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]-piperazin-1-yloxy}-1-β-d-glucuronic acid (3) was also observed in biological samples from humans and animals and the first organic synthesis and structural identification of this metabolite is also reported. Treatment of the glucuronide metabolites 3 and 4 with β-glucuronidase gave mainly the expected hydrolysis product, the hydroxyl amine 4-[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]-piperazin-1-ol (2).

Pharmacological in vitro characterization of the arecoline bioisostere, Lu 25-109-T, a muscarinic compound with M1-Agonistic and M2/M3-antagonistic properties

The arecoline bioisostere, Lu 25‐109‐T, displays a pharmacological profile of a partial muscarinic agonist with a several‐fold higher affinity for cortical M1 receptors than for brain stem M2 receptors and salivary glands M3 receptors. The compound is selective for muscarinic receptors as it shows no or only low affinity for other receptor types. In functional assays Lu 25‐109‐T behaves as a partial agonist at the guinea pig ileum (M1/M2/M3), at the rat superior cervical ganglion (M1 and at cells transfected with cloned human ml muscarinic receptors and as an antagonist at guinea pig left atrium (M2) and cultured cerebellar granule cells (M3). Lu 25‐109‐T readily passes the blood‐brain barrier in mice and has a bioavailability of 42% at oral administration although with a short half‐life (t½=41 min).

Radiolabelling and PET brain imaging of the α1-adrenoceptor antagonist Lu AE43936

Cerebral α₁-adrenoceptors are a common target for many antipsychotic drugs. Thus, access to positron emission tomography (PET) brain imaging of α₁-adrenoceptors could make important contributions to the understanding of psychotic disorders as well as to the pharmacokinetics and occupancy of drugs targeting the α₁-adrenoceptors. However, so far no suitable PET radioligand has been developed for brain imaging of α₁-adrenoceptors. Here, we report the synthesis of both enantiomers of the desmethyl precursors of the high affinity α₁-adrenoceptor ligand (1). The two enantiomers of 1 were subsequently [¹¹C] radiolabelled and evaluated for brain uptake and binding by PET imaging in Danish Landrace pigs. (S)-[¹¹C]-1 and (R)-[¹¹C]-1 showed very limited brain uptake. Pre-treatment with cyclosporine A (CsA) resulted in a large increase in brain uptake, indicating that (R)-[¹¹C]-1 is a substrate for active efflux-transporters. This was confirmed in Madin Darby canine kidney (MDCK) cells overexpressing permeability glycoprotein (Pgp). In conclusion, the limited brain uptake of both (S)-[¹¹C]-1 and (R)-[¹¹C]-1 in the pig brain necessitates the search for alternative radioligands for in vivo PET brain imaging of α₁-adrenoceptors.

Discriminative stimulus properties of the muscarinic receptor agonists Lu 26-046 and O-Me-THPO in rats: evidence for involvement of different muscarinic receptor subtypes.

The discriminative cues induced by the muscarinic receptor agonists Lu 26-046 ((-)-7-methyl-3(2-propynyloxy)-4,5,6,7-tetrahydroisothiazolo [4,5-c]pyridine ) and O-Me-THPO (3-methoxy-4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine) were investigated. The results were compared with those obtained for the binding profiles of these agonists at central muscarinic receptors and with results concerning their functional effects at peripheral muscarinic receptors in vitro. Lu 26-046 had preferential affinity for M1 versus M2 receptors (Ki index [3H]quinuclidinyl benzilate ([3H]QNB/[3H]pirenzepine 4.2) and had partial agonistic activity at M1 and M2 receptors in rat superior cervical ganglion and guinea pig left atrim, respectively. A weak antagonistic effect at M3 receptors in guinea pig ileum was observed. O-Me-THPO had non-selective agonistic effects at peripheral M1, M2 and M3 receptors and had a slight preference for central M2 receptors in binding experiments (M2/M1 index 0.31). Lu 26-046 dose dependently substituted for Lu 26-046 and partially substituted for O-Me-THPO in rats trained to discriminate Lu 26-046 and O-Me-THPO from saline, respectively. The (+)-enantiomer of Lu 26-046, Lu 26-047, had weak partial M1 agonistic activity and M2/M3 antagonistic effects at peripheral receptors. Lu 26-047 also had a high M2/M1 index (9.3) in binding experiments. Lu 26-047 substituted for Lu 26-046, but preferentially inhibited the effect of O-Me-THPO. Pilocarpine had a preferential effect in Lu 26-046-trained rats, while oxotremorine and arecoline had preferential effects in O-Me-THPO-trained rats. Large increases in latency times or a disruption of responding was generally observed. These compounds were full agonists at peripheral M1, M2 and M3 receptors. The muscarinic receptor antagonist scopolamine antagonized the effect of O-Me-THPO and partially inhibited the effect of Lu 26-046. Scopolamine partially substituted for Lu 26-046. The quaternary muscarinic receptor agonist N-methyl atropine had no effect, indicating that the cues are mediated by central muscarinic receptors. It is suggested that the discriminative cues of Lu 26-046 and O-Me-THPO are preferentially mediated by central M1 (partial) and M2 receptor stimulation, respectively. The role of central M3 receptors is not known.

Biological conversion of aripiprazole lauroxil − An N-acyloxymethyl aripiprazole prodrug

N-acyloxyalkylation of NH-acidic compounds can be a prodrug approach for e.g. tertiary or some N-heterocyclic amines and secondary amides and have the potential to modify the properties of the parent drug for specific uses, for example its physicochemical, pharmacokinetic or biopharmaceutical properties. Aripiprazole lauroxil was prepared as a model compound for such prodrugs and its bioconversion was investigated both in vitro and in vivo. Theoretically, N-acyloxyalkyl derivates of NH-acid compounds undergo a two-step bioconversion into the parent NH-acidic drug through an N-hydroxyalkyl intermediate. However, to our knowledge no published studies have investigated the formation of an intermediate in vivo. In the present study, it was demonstrated that the assumed N-hydroxymethyl intermediate was readily observed both in vitro and in vivo. In vivo, the observed plasma concentration of the intermediate was at the same level as the drug (aripiprazole). When prodrug intermediates are formed, it is important to make a proper pharmacological, pharmacokinetic and toxicological evaluation of the intermediates to ensure patient safety; however, several challenges were identified when testing an N-acyloxyalkyl prodrug. These included the development of a suitable bioanalytical method, the accurate prediction of prodrug bioconversion and thereby the related pharmacokinetics in humans and the toxicological potential of the intermediate.

Discovery and structure-activity relationship of 1,3-cyclohexyl amide derivatives as novel mGluR5 negative allosteric modulators.

A novel series of trans-1,3-cyclohexyl diamides was discovered and characterized as mGluR5 negative allosteric modulators (NAMs) lacking an alkyne moiety. Conformational constraint of one of the amide bonds in the diamide template led to a spirooxazoline template. A representative compound (24d) showed good in vitro potency, high CNS penetration and, upon subcutaneous dosing, demonstrated efficacy in the mouse marble burying test, generally used as indicative of potential anxiolytic activity.

Synthesis of Two d‐Glucosamine Derived 3,4‐Epoxides as Potential Scaffolds for Combinatorial Chemistry

Combinatorial chemistry allows the synthesis of libraries of compounds by combination of building blocks or by combinatorial elaboration of a central scaffold. Carbohydrates hold great promise as scaffolds due to their high degree of functionalization, relative conformational rigidity, commercial availability of many stereoisomeric forms, and their well-described chemistry. Hirschmann, Nicolaou, Smith, and their coworkers pioneered the use of carbohydrates as scaffolds in their design and synthesis of b-D-glucose derived non-peptide peptidomimetics of the peptide hormone

Muscarinic agonists. Syntheses and structure—activity relationships of bicyclic isoxazole ester bioisosteres of norarecoline

Summary ( RS )-3-Methoxy-8-methyl-5,6,7,8-tetrahydro-4 H -isoxazolo[4,5- c ]azepine ( O ,8-di-Me-THAO, 2c) and ( RS )-8-methyl-3-propargyloxy-5,6,7,8-tetrahydro-4 H -isoxazolo[4,5- c ]azepine (8-Me- O -propargyl-THAO, 2d ) have been synthesized and evaluated as muscarinic receptor ligands in receptor binding assays and in in vitro functional assays. The corresponding compounds without methyl groups at C-8, ie O -Me-THAO ( 2a ) and O -propargyl-THAO ( 2b ), have previously been shown to exhibit muscarinic agonistic profiles with very little discrimination between M 1 and M 2 -receptor sites. Based on functional assays, 2c and 2d were found to be less efficacious than 2a and 2b , respectively, and 2d proved to be an M 1 -selective partial agonist. The affinity and M 1 efficacy of 2c and 2d were comparable to those of the corresponding six-membered ring analogues, ( RS ) -3-methoxy-7-methyl-4,5,6,7-tetrahydroisoxazolo [4,5- c ]pyridine (0,7-di-Me-THPO, 1c ) and ( RS )-7-methyl-3-propargyloxy-4,5,6,7-tetrahydroisoxazolo[4,5- c ]pyridine (7-Me- O -propargyl-THPO, 1d ). However, neither compound 1c nor compound 1d displayed M 1 selectivity. In summary, within this class of bicyclic muscarinic agonists, replacement of 3-methoxy by 3-propargyloxy groups generally increases muscarinic affinity without affecting the efficacy at M 1 receptors significantly. Introduction of a methyl group into the saturated ring at the position α to the C-5 of the isoxazole ring (α-position) leads to compounds exerting lower efficacy and, in the case of compound 2d , an increased selectivity with respect to M 1 agonism.

Tying up Nicotine: New Selective Competitive Antagonist of the Neuronal Nicotinic Acetylcholine Receptors.

Conformational restriction of the pyrrolidine nitrogen in nicotine by the introduction of an ethylene bridge provided a potent and selective antagonist of the α4β2-subtype of the nicotinic acetylcholine receptors. Resolution by chiral SFC, pharmacological characterization of the two enantiomers, and determination of absolute configuration via enantioselective synthesis showed that the pharmacological activity resided almost exclusively in the (R)-enantiomer.