Antipsychotic Benzamides Amisulpride and LB-102 Display Polypharmacy as Racemates, S Enantiomers Engage Receptors D2 and D3, while R Enantiomers Engage 5-HT7.

Benzamide antipsychotics such as amisulpride are dosed as racemates though efficacy is assumed to be mediated through S enantiomer binding to D2 receptors. At prescribed doses, the benzamides likely display polypharmacy since brain exposure should be sufficient to engage the 5-HT7 receptors, as well. Curiously, the studies herein reveal that racemic dosing is required to engage both targets since the D2 receptor has an almost 40-fold selectivity for the S enantiomer, while the 5-HT7 receptor has greater than 50-fold preference for the R enantiomer.

Introduction

There is a strong preference for new drugs to be developed as single enantiomers to avoid risks associated with dosing a distomer (inactive enantiomer) along with its eutomer (active enantiomer) unless there is a compelling reason to use the racemic mixture.[1,2] As such, when developing a new drug with a chiral center, it has become a common practice to identify the active enantiomer and to develop it as a chirally pure compound. The regulatory environment of the past was less stringent, leading to a number of racemic drugs on the market in the US and Europe.[1] For example, many members of the benzamide class of antipsychotics exemplified by the compounds listed in Figure are dosed as racemates.

Figure 1

Representative benzamide antipsychotics. Top panels: plots of D2 apparent affinity for dopamine in the presence of varied amisulpride concentration (left) or varied LB-102, LB-103, or LB-104 concentration (right). Bottom panels: plots of 5-HT7 apparent affinity for serotonin in the presence of varied (S), (R), or (rac) amisulpride concentration (left) or varied LB-102, LB-103, or LB-104 concentration (right).

Benzamide antipsychotics were developed to engage the brain dopamine D2 and D3 receptors (referred to collectively as D2/3 in the text) and fall into the category of atypical antipsychotics.[3] Though never submitted for regulatory approval in the United States, numerous clinical trials demonstrate that the benzamide amisulpride is one of the most effective antipsychotics on the market in Europe.[4] In addition to the antipsychotic activity of amisulpride, there are many reports of its antidepressant activity in schizophrenia and other indications.[5−7] The work of Abbas et al., consisting of rodent in vivo studies, as well as the examination of a panel of off-target receptors, identified the serotonin 5-HT7 receptor as the likely mediator of amisulpride’s antidepressant activity.[8]

Polypharmacology occurs when one drug engages multiple targets that each contribute to a disease and is distinct from polypharmacy where multiple drugs are used to engage these targets.[9] As is the case with the benzamides, often, polypharmacology is a serendipitous outcome. Nevertheless, when discovered, polypharmacology is frequently invoked to explain the unusually high efficacy observed with such drugs. Adding to the complexity surrounding amisulpride’s efficacy is the racemic nature of the drug. The chiral preference of the D2/3 receptors was determined in 2001 when Castelli et al. reported that the S enantiomer of amisulpride bound D2/3 nearly 40-fold more potently than the R enantiomer.[10] The enantiomeric preference of the 5-HT7 receptor for amisulpride is reported in this work.

LB Pharmaceuticals is developing a new generation of N-alkylated benzamide antipsychotics. Promising preclinical results were obtained with LB-102, an N-methylated analogue of amisulpride, and with LB-103, the N-methylated S enantiomer of amisulpride[11,12] (Figure ). The 5-HT7 receptor stereo preference played a key role in deciding which asset to advance. If the 5-HT7 receptor, like the D2/3 receptors, displayed a preference for the S enantiomer, then the logical choice would be to advance chirally pure LB-103. Conversely, if the 5-HT7 receptor was selective for the R enantiomer (LB-104), then to harness the benzamide polypharmacology, we will advance the racemic compound LB-102, which being two distinct compounds would define the effect as polypharmacy rather than polypharmacology.

Figure 2

N-Methylated benzamides under development.

Results and Discussion

Receptor binding studies were conducted in a cell-based assay containing overexpressed target receptors and a modified cyclic nucleotide-gated ion channel.[13]

Receptor engagement by the antagonists was determined from an analysis of observed response to a concentration matrix of natural ligands, dopamine or serotonin, versus a gradient of concentrations of antagonists. The apparent affinity Kd(apparent) of the natural ligand was calculated at fixed concentrations of the antagonist and then replotted as a function of antagonist concentration (Figure ). These studies are ligand displacement assays and conform to the Cheng–Prusoff relationship for competitive inhibition.[14] When (R), (S), and (rac) compounds were examined against the receptor, eqs –3 were fit globally to the results. Kd refers to the binding affinity of the natural ligand and KI refers to the binding affinity of the antagonistAs seen in Figure , the displacement assays are consistent with a competitive Cheng–Prusoff relationship since they produce the expected linear relationships between apparent ligand affinities and antagonist concentrations. The antagonist binding affinities are presented in Table . The D2 receptor prefers the S benzamide enantiomer having an almost 40-fold preference for LB-103 v LB-104, comparable to the amisulpride preferences reported by Castelli et al.[10] As well, within experimental uncertainty, LB-102 and amisulpride bind to the D2 receptor with a Kd of 1 nM. In contrast, the 5-HT7 receptor has a striking 50-fold preference for the R enantiomers of the benzamides. The racemic benzamide affinity for the 5-HT7 receptor is approximately 30-fold weaker than its D2 receptor affinity though potentially still pharmacologically relevant.

Figure 3

Natural ligand displacement by antagonists.

Table 1

Receptor Binding Affinitya

antagonist	D2KI (nM)	5-HT7 KI (nM)
amisulpride (S)	n.d.	900 ± 1300
amisulpride (rac)	1.1 ± 0.12	44 ± 3
amisulpride (R)	n.d.	22 ± 1.5
LB-102	0.82 ± 0.02	31 ± 1
LB-103	0.4 ± 0.04	>1000
LB-104	14.4 ± 2.2	15.6 ± 0.9

n.d. not determined.

Amisulpride effectively moderates schizophrenia’s negative symptoms at moderate daily doses (50–300 mg) and the positive symptoms at higher daily doses (800–1600 mg).[15] The human pharmacokinetics of amisulpride are dose-linear over its therapeutic dose range, and its plasma PK and brain D2/3 occupancies are reported.[15,16] PK–D2/3 occupancy models of amisulpride and LB-102 were generated[12] by combining reports on amisulpride human PK,[15,16] D2/3 receptor occupancy,[16] rat dose–D2/3 receptor occupancy and behavioral studies,[17] and in-house rat PK studies. The absence of in vivo 5-HT7 receptor occupancy studies in animals or humans limits our ability to reliably model 5-HT7 in vivo occupancy since in vitro binding affinities rarely translate well to in vivo occupancies. Our previous work predicts that an 800 mg dose of amisulpride will produce a peak brain concentration of 20 ng/g, 10 h post dose, which provides a 90% D2/3 occupancy. Our work determined an effective human brain D2/3 EC50 of 1.6 ng/g. Extrapolating the in vitro differences in potency between D2 and 5-HT7 to approximate in vivo 5-HT7 EC50 predicts a 5-HT7 occupancy of around 30% at 800 mg. Abbas reports an amisulpride KI of 11.5 nM v LSD, which would translate to a 50% occupancy at an 800 mg dose. Reliably modeling 5-HT7 occupancy in humans will require a competing PET ligand study, as has been done with the D2/3 receptors.

Conclusions

In the development of new pharmaceuticals, racemates are avoided when possible and new drugs are preferentially developed in enantiomerically pure form. As described in this work, the benzamide antipsychotics present an interesting challenge to the developmental process. The benzamides display polypharmacy if dosed as a racemate: antipsychotic activity is provided by targeting D2/3 receptors with the S enantiomer, while antidepressant effects are provided by targeting the 5-HT7 receptor with the R enantiomer. Based on the findings in this report, racemic LB-102 will be advanced to the clinic rather than the enantiomerically pure S LB-103.

Experimental Section

Compounds: amisulpride (rac) and (S) enantiomers were purchased from Toronto Research Chemicals (Toronto, ON); amisulpride (R), LB-102 (rac), LB-103 (S), and LB-104 (R) were prepared by Kalexsyn (Kalamazoo, MI): briefly, racemic amisulpride (and individual enantiomers) was suspended in N,N-dimethylformamide dimethyl acetal and stirred at 90 °C for 2 h; the mixture was cooled to ambient temperature, NaBH4 was added portionwise, and the mixture was stirred for 1 h. The organic phase was removed under reduced pressure, and the residue was extracted with dichloromethane, washed with brine, dried, and filtered, and the solvent was removed under reduced pressure. Purification was achieved by column chromatography followed by recrystallization from acetone.

Compound structural integrities were determined by proton NMR. The purities were assessed using HPLC. All compounds possess a purity of at least 95%. Details are available in the Supporting Information.

Receptor binding studies were conducted by CODEX BIOSOLUTIONS using their ACTOne system assay procedures, and details are provided on their website.[13] In brief, ACTOne cell system contains a modified cyclic nucleotide-gated ion channel as a biosensor of cAMP activity in live cells. The channel responds to increases or decreases in intracellular cAMP levels by coordinately altering calcium flux, which is measured with a calcium-sensitive dye.[13] Cell lines were developed containing overexpressed D2 dopamine receptors or 5-HT7 serotonin receptors.

Assays were conducted in duplicate in 384-well plates. The concentration matrix consisted of five concentrations of the antagonist at 3-fold dilutions (and a 0 concentration) versus six concentrations of natural ligand also at 3-fold dilutions. Natural ligand binding (dopamine for D2 and 5-HT for 5-HT7) was determined from the response curves of natural ligand at a fixed antagonist concentration using a four-parameter binding equationwhere max = response at a saturating ligand concentration, min = response in the absence of ligand, Kdapparent = ligand concentration producing half the maximum signal, and n = the Hill coefficient. Binding affinities for the antagonists were determined from fits of Kdapparent versus antagonist concentration using eqs –3.